Encoded solid supports for biological processing and assays using same

ABSTRACT

Combinations, called matrices with memories, of matrix materials with remotely addressable or remotely programmable recording devices that contain at least one data storage unit are provided. The matrix materials are those that are used in as supports in solid phase chemical and biochemical syntheses, immunoassays and hybridization reactions. The matrix materials may additionally include fluophors or other luminescent moieties to produce luminescing matrices with memories. The data storage units are non-volatile antifuse memories or volatile memories, such as EEPROMS, DRAMS or flash memory. By virtue of this combination, molecules and biological particles, such as phage and viral particles and cells, that are in proximity or in physical contact with the matrix combination can be labeled by programming the memory with identifying information and can be identified by retrieving the stored information. Combinations of matrix materials, memories, and linked molecules and biological materials are also provided. The combinations have a multiplicity of applications, including combinatorial chemistry, isolation and purification of target macromolecules, capture and detection of macromolecules for analytical purposes, selective removal of contaminants, enzymatic catalysis, cell sorting, drug delivery, chemical modification and other uses. Methods for electronically tagging molecules, biological particles and matrix support materials, immunoassays, receptor binding assays, scintillation proximity assays, non-radioactive proximity assays, and other methods are also provided.

RELATED APPLICATIONS

For U.S. national purposes, this application is a continuation-in-partof U.S. application Ser. No. 08/DKT302B, filed Apr. 2, 1996, entitledREMOTELY PROGRAMMABLE MATRICES WITH MEMORIES AND USES THEREOF”, byMichael P. Nova, Andrew E. Senyei, Zahra Parandoosh and Gary S. David,which application is a continuation-in-part of U.S. application Ser. No.08/567,746, filed Dec. 5, 1995, entitled REMOTELY PROGRAMMABLE MATRICESWITH MEMORIES AND USES THEREOF”, by Michael P. Nova, Andrew E. Senyei,Zahra Parandoosh and Gary S. David, which application is acontinuation-in-part of U.S. application Ser. No. 08/538,387, filed Oct.3, 1995, entitled “REMOTELY PROGRAMMABLE MATRICES WITH MEMORIES”,Michael P. Nova, Andrew E. Senyei, and Gary S. David, which in turn is acontinuation-in-part of U.S. application Ser. Nos. 08/480,147,08/484,486, 08/484,504, 08/480,196 and 08/473,660, each filed Jun. 7,1995, and each entitled, “REMOTELY PROGRAMMABLE MATRICES WITH MEMORIES”.

This application is also a continuation-in-part of U.S. application Ser.No. 08/538,387, and a continuation-in-part of each of U.S. applicationSer. Nos. 08/480,147, 08/484,486, 08/484,504, 08/480,196, 08/473,660,and 08/428,662, filed Apr. 25, 1995, by Michael P. Nova and Andrew E.Senyei, entitled, “REMOTELY PROGRAMMABLE MATRICES WITH MEMORIES”. Eachof U.S. application Ser. Nos. 08/dkt302, 08/567,746, 08/538,387,08/480,147, 08/484,486, 08/484,504, 08/480,196 and 08/473,660 is acontinuation-in-part of U.S. application Ser. No. 08/428,662.

The subject matter of each of U.S. application Ser. Nos. 08/DKT302B,08/567,746, 08/538,387, 08/480,147, 08/484,486, 08/484,504, 08/480,196,08/473,660 and 08/428,662 is incorporated herein by reference in itsentirety. The subject matter of each of U.S. application Ser. Nos.08/379,923 and 08/322,644 also is incorporated herein its entirety.

FIELD OF THE INVENTION

The present invention relates to the application of data storagetechnology to molecular tracking and identification and to biological,chemical, immunological and biochemical assays.

BACKGROUND OF THE INVENTION

Drug discovery relies on the ability to identify compounds that interactwith a selected target, such as cells, an antibody, receptor, enzyme,transcription factor or the like. Traditional drug discovery relied oncollections or “libraries” obtained from proprietary databases ofcompounds accumulated over many years, natural products, fermentationbroths, and rational drug design. Recent advances in molecular biology,chemistry and automation have resulted in the development of rapid, Highthroughput screening (HTS) protocols to screen these collection. Inconnection with HTS, methods for generating molecular diversity and fordetecting, identifying and quantifying biological or chemical materialhave been developed. These advances have been facilitated by fundamentaldevelopments in chemistry, including the development of highly sensitiveanalytical methods, solid state chemical synthesis, and sensitive andspecific biological assay systems.

Analyses of biological interactions and chemical reactions, however,require the use of labels or tags to track and identify the results ofsuch analyses. Typically biological reactions, such as binding,catalytic, hybridization and signaling reactions, are monitored bylabels, such as radioactive, fluorescent, photoabsorptive, luminescentand other such labels, or by direct or indirect enzyme labels. Chemicalreactions are also monitored by direct or indirect means, such as bylinking the reactions to a second reaction in which a colored,fluorescent, chemiluminescent or other such product results. Theseanalytical methods, however, are often time consuming, tedious and, whenpracticed in vivo, invasive. In addition, each reaction is typicallymeasured individually, in a separate assay. There is, thus, a need todevelop alternative and convenient methods for tracking and identifyinganalytes in biological interactions and the reactants and products ofchemical reactions.

Combinatorial Libraries

The provision and maintenance of compounds to support HTS have becomecritical. New and innovative methods for the lead generation and leadoptimization have emerged to address this need for diversity. Amongthese methods is combinatorial chemistry, which has become a powerfultool in drug discovery and materials science. Methods and strategies forgenerating diverse libraries, primarily peptide- and nucleotide-basedoligomer libraries, have been developed using molecular biology methodsand/or simultaneous chemical synthesis methodologies [see, e.g., Doweret al. (1991) Annu. Rep. Med. Chem. 26:271-280; Fodor et al. (1991)Science 251:767-773; Jung et al. (1992) Angew. Chem. Ind. Ed. Engl.31:367-383; Zuckerman et al. (1992) Proc. Natl. Acad. Sci. USA89:4505-4509; Scott et al. (1990) Science 249:386-390; Devlin et al.(1990) Science 249:404-406; Cwirla et al. (1990) Proc. Natl. Acad. Sci.USA 87:6378-6382; and Gallop et al. (1994) J. Medicinal Chemistry37:1233-1251]. The resulting combinatorial libraries potentially containmillions of pharmaceutically relevant compounds and that can be screenedto identify compounds that exhibit a selected activity.

The libraries fall into roughly three categories:fusion-protein-displayed peptide libraries in which random peptides orproteins are presented on the surface of phage particles or proteinsexpressed from plasmids; support-bound synthetic chemical libraries inwhich individual compounds or mixtures of compounds are presented oninsoluble matrices, such as resin beads [see, e.g., Lam et al. (1991)Nature 354:82-84] and cotton supports [see, e.g., Eichler et al. (1993)Biochemistry 32:11035-11041]; and methods in which the compounds areused in solution [see, e.g., Houghten et al. (1991) Nature 354:84-86,Houghten et al. (1992) BioTechniques 313:412-421; and Scott et al.(1994) Curr. Opin. Biotechnol. 5:40-48]. There are numerous examples ofsynthetic peptide and oligonucleotide combinatorial libraries. Thepresent direction in this area is to produce combinatorial librariesthat contain non-peptidic small organic molecules. Such libraries arebased on either a basis set of monomers that can be combined to formmixtures of diverse organic molecules or that can be combined to form alibrary based upon a selected pharmacophore monomer.

There are three critical aspects in any combinatorial library: (i) thechemical units of which the library is composed; (ii) generation andcategorization of the library, and (iii) identification of librarymembers that interact with the target of interest, and trackingintermediary synthesis products and the multitude of molecules in asingle vessel.

The generation of such libraries often relies on the use of solid phasesynthesis methods, as well as solution phase methods, to producecollections containing tens of millions of compounds that can bescreened in diagnostically or pharmacologically relevant in vitro assaysystems. In generating large numbers of diverse molecules by stepwisesynthesis, the resulting library is a complex mixture in which aparticular compound is present at very low concentrations, so that it isdifficult or impossible to determine its chemical structure. Variousmethods exist for ordered synthesis by sequential addition of particularmoieties, or by identifying molecules based on spatial positioning on achip. These methods are cumbersome and ultimately impossible to apply tohighly diverse and large libraries. Identification of library membersthat interact with a target of interest, and tracking intermediarysynthesis products and the multitude of molecules in a single vessel isalso a problem.

High Throughput Screening

In addition, exploitation of this diversity requires development ofmethods for rapidly screening compounds. Advances in instrumentation,molecular biology and protein chemistry and the adaptation ofbiochemical activity screens into microplate formats, has made itpossible to screen of large numbers of compounds. Also, because compoundscreening has been successful in areas of significance for thepharmaceutical industry, high throughput screening (HTS) protocols haveassumed importance. Presently, there are hundreds of HTS systemsoperating throughout the world, which are used, not only for compoundscreening for drug discovery, but also for immunoassays, cell-basedassays and receptor-binding assays.

An essential element of high throughput screening for drug discoveryprocess and areas in which molecules are identified and tracked, is theability to extract the information made available during synthesis andscreening of a library, identification of the active components ofintermediary structures, and the reactants and products of assays. Whilethere are several techniques for identification of intermediary productsand final products, nanosequencing protocols that provide exactstructures are only applicable on mass to naturally occurring linearoligomers such as peptides and amino acids. Mass spectrographic [MS]analysis is sufficiently sensitive to determine the exact mass andfragmentation patterns of individual synthesis steps, but complexanalytical mass spectrographic strategies are not readily automated norconveniently performed. Also, mass spectrographic analysis provides atbest simple connectivity information, but no stereoisomeric information,and generally cannot discriminate among isomeric monomers. Anotherproblem with mass spectrographic analysis is that it requires purecompounds; structural determinations on complex mixtures is eitherdifficult or impossible. Finally, mass spectrographic analysis istedious and time consuming. Thus, although there are a multitude ofsolutions to the generation of libraries and to screening protocols,there are no ideal solutions to the problems of identification, trackingand categorization.

These problems arise in any screening or analytical process in whichlarge numbers of molecules or biological entities are screened. In anysystem, once a desired molecules has been isolated, it must beidentified. Simple means for identification do not exist. Because of theproblems inherent in any labeling procedure, it would be desirable tohave alternative means for tracking and quantitating chemical andbiological reactions during synthesis and/or screening processes, andfor automating such tracking and quantitating.

Therefore, it is an object herein to provide methods for identification,tracking and categorization of the components of complex mixtures ofdiverse molecules. It is also an object herein to provide products forsuch identification, tracking and categorization and to provide assays,diagnostics and screening protocols that use such products. It is ofparticular interest herein to provide means to track and identifycompounds and to perform HTS protocols.

SUMMARY OF THE INVENTION

Combinations of matrix materials with programmable data storage orrecording devices, herein referred to as memories, and assays usingthese combinations are provided. These combinations are referred toherein as matrices with memories. By virtue of this memory with matrixcombination, molecules, such as antigens, antibodies, ligands, proteinsand nucleic acids, and biological particles, such as phage and viralparticles and cells, that are associated with, such as in proximity toor in physical contact with the matrix combination, can beelectromagnetically tagged by programming the memory with datacorresponding to identifying information. Programming and reading thememory is effected remotely, preferably using electromagnetic radiation,particularly radio frequency or radar. Memories may also be remote fromthe matrix, such as instances in which the memory device is precodedwith a mark or identifier or the matrix is encoded with a bar code. Theidentity [i.e., the mark or code] of each device is written to a memory,which may be a computer or a piece of paper or any recording device, andinformation associated with each matrix is stored in the remote memoryand linked to the code or other identifier.

The molecules and biological particles that are associated with thematrix combination, such as in proximity to or in physical contact orwith the matrix combination, can be identified and the results of theassays determined by retrieving the stored data points from thememories. Querying the memory will identify associated molecules orbiological particles that have reacted.

In certain embodiments of the matrices with memories, reactions, assaysand other events or external parameters, such as temperature and/or pH,can be monitored because occurrence of a reaction or an event can bedetected and such detection sent to the recording device and recorded inthe memory.

The combinations provided herein thus have a multiplicity ofapplications, including combinatorial chemistry, isolation andpurification of target macromolecules, capture and detection ofmacromolecules for analytical purposes, high throughput screening,selective removal of contaminants, enzymatic catalysis, drug delivery,chemical modification, information collection and management and otheruses. These combinations are particularly advantageous for use inmultianalyte analyses, assays in which a electromagnetic signal isgenerated by the reactants or products in the assay, for use inhomogeneous assays, and for use in multiplexed protocols.

In preferred embodiments, these matrix with memory combinations contain(i) a miniature recording device that includes one or more programmabledata storage devices [memories] that can be remotely read and inpreferred embodiments also remotely programmed; and (ii) a matrix, suchas a particulate support used in chemical syntheses.

The matrix materials [matrices] are any materials that are routinelyused in chemical and biochemical synthesis. The matrix materials aretypically polymeric materials that are compatible with chemical andbiological syntheses and assays, and include, glasses, silicates,celluloses, polystyrenes, polysaccharides, polypropylenes, sand, andsynthetic resins and polymers, including acrylamides, particularlycross-linked polymers, cotton, and other such materials. The matricesmay be in the form of particles or may be continuous in design, such asa test tube or microplate, 96 well or 384 well or higher density formatsor other such microplates and microtiter plates. The matrices maycontain one or a plurality of recording devices. For example, each wellor selected wells in the microplate include a memory device in contacttherewith or embedded therein. The plates may further contain embeddedscintillant or a coating of scintillant [such as FlashPlate™, availablefrom DuPont NEN®, and plates available from Packard, Meriden, Conn.].Automated robotic protocols will incorporate such plates for automatedmultiplexing [performing a series of coupled synthetic and processingsteps, typically, though not necessarily on the same platform, i.e.coupling of the chemistry to the biology] including one or more of thefollowing, synthesis, preferably accompanied by writing to the linkedmemories to identify linked compounds, screening, including usingprotocols with matrices with memories, and compound identification byquerying the memories of matrices associated with the selectedcompounds.

The matrices are either particulate of a size that is roughly about 1 to20 mm³ [or 1-20 mm in its largest dimension], preferably about 10 mm³ orsmaller, preferably 1 mm³ or smaller, or a continuous medium, such as amicrotiter plate, or other multi-well plate, or plastic or other solidpolymeric vial or glass vial or catheter-tube [for drug delivery] orsuch container or device conventionally used in chemistry and biologicalsyntheses and reactions. In instances in which the matrix is continuous,the data storage device [memory] may be placed in, on, or under thematrix medium or may be embedded in the material of the matrix.

In embodiments herein in which the matrices with memories are used inassays, such as scintillation proximity assays [SPA], FP [fluorescencepolarization] assays, FET [fluorescent energy transfer] assays, FRET[fluorescent resonance energy transfer] assays and HTRF [homogeneoustime-resolved fluorescence] assays, the matrices may be coated with,embedded with or otherwise combined with or in contact with assaymaterial, such as scintillant, fluophore or other fluorescent label. Theresulting combinations are called luminescing memories with matrices.When used in SPA formats they are referred to as scintillating matriceswith memories and when used in non-radioactive energy transfer formats[such as HTRF] they are referred to as fluorescing memories withmatrices.

The recording device is preferably a miniature device, typically lessthan 10-20 mm³ [or 10-20 mm in its largest dimension] in size,preferably smaller, such as 1 to 5 mm, that includes at least one datastorage unit that includes a remotely programmable and remotelyreadable, preferably non-volatile, memory. This device with remotelyprogrammable memory is in proximity to, associated with or in contactwith the matrix. In particular, the recording device includes a memorydevice, preferably having memory means, preferably non-volatile, forstoring a plurality of data points and means for receiving a transmittedsignal that is received by the device and for causing a data pointcorresponding to the data signal to be permanently stored within thememory means. If needed, the recording device further includes a shell[coating] that is non-reactive with and impervious to any processingsteps or solutions in which the combination of matrix with recordingdevice [matrix with memory] is placed, and that is transmissive of reador write signals transmitted to the memory. The device may also includeat least one support matrix disposed on an outer surface of the shellfor retaining molecules or biological particles. The shell and supportmatrix may be the same. In such instances, the shell must be treated orderivatized such that molecules, particularly amino acids and nucleicacids, can be linked, preferably either electrostatically or covalently,thereto. Thus, a transponder enclosed in plastic, must be furthertreated or coated to render it suitable for linkage of the molecule orbiological particle.

The data storage device or memory is programmed with or encoded withinformation that identifies molecules or biological particles, either bytheir process of preparation, their identity, their batch number,category, physical or chemical properties, combinations of any of suchinformation, or other such identifying information. The molecules orbiological particles are in physical contact, direct or indirect, or inproximity with the matrix, which in turn is in physical contact or inthe proximity of the recording device that contains the data storagememory. The molecule or biological particle may also be associated, suchthat a molecule or biological particle that had been linked to or inproximity with a matrix with memory may be identified [i.e., althoughthe matrix particle and biological particle or molecule are not linkedor in proximity, the identify of the matrix that had been linked to themolecule or particle is known]. Typically, the matrix is on the surfaceof the recording device and the molecules and biological particles arein physical contact with the matrix material. In certain embodiments,the memory device may be linked to or in proximity to more than onematrix particle.

The data storage device or memory can also be programmed by virtue of areaction in proximity to or in the vicinity of the matrix with memory.In particular, the recording devices include memories and alsoadditional components that detect occurrence of external events or tomonitor the status of external parameters, such as EM emissions, changesin temperature or pH, ion concentrations and other such solutionparameters. For example, recording devices include memories and alsoinclude a photodetector can detect the occurrence of fluorescence orother optical emission. Coupling this emission with an amplifier andproviding a voltage to permit data storage in the matrix with memoryduring the reaction by way of, for example an RF signal transmitted toand received by an antenna/rectifier combination within the data storagedevice or providing voltage sufficient to write to memory from a battery[see, e.g., U.S. Pat. No. 5,350,645 and U.S. Pat. No. 5,089,877],permits occurrence of the emission to be recorded in the memory.

The recording device [containing the memory] is associated with thememory. Typically, the recording device is coated with at least onelayer of material, such as a protective polymer or a glass, includingpolystyrene, heavy metal-free glass, plastic, ceramic, and may be coatedwith more than one layers of this and other materials. For example, itmay be coated with a ceramic or glass, which is then coated with orlinked to the matrix material. Alternatively, the glass or ceramic orother coating may serve as the matrix. In other embodiments therecording device and the matrix material are in proximity, such as in acontainer of a size approximately that of the device and matrixmaterial. In yet other embodiments the recording device and matrixmaterial are associated, such that the molecule or biological particlethat was linked to the matrix or that was in proximity thereto may beidentified.

The matrix combinations [the memories with matrices], thus, contain amatrix material, typically in particulate form, in physical contact witha tiny device containing one or more remotely programmable data storageunits [memories]. Contact can be effected by placing the recordingdevice with memory on or in the matrix material or in a solution that isin contact with the matrix material or by linking the device, either bydirect or indirect covalent or non-covalent interactions, chemicallinkages or by other interactions, to the matrix.

For example, such contact is effected chemically, by chemically couplingthe recording device with memory to the matrix, or physically by coatingthe recording device with the matrix material or another material, byphysically inserting or encasing the device in the matrix material, byplacing the device onto the matrix or by any other means by which thedevice can be placed in contact with or in proximity to the matrixmaterial. The contact may be direct or indirect via linkers. The contactmay be effected by absorption or adsorption.

Since matrix materials have many known uses in conjunction withmolecules and biological particles, there are a multitude of methodsknown to artisans of skill in this art for linking, joining orphysically contacting the molecule or biological particle with thematrix material. In some embodiments, the recording device with datastorage unit is placed in a solution or suspension of the molecule orbiological particle of interest. In some of such instances, thecontainer, such as the microtiter plate or test tube or other vial, isthe matrix material. The recording device is placed in or on the matrixor is embedded, encased or dipped in the matrix material or otherwiseplace in proximity by enclosing the device and matrix material in asealed pouch or bag or container [MICROKAN™] fabricated from,preferably, porous material, such as teflon or polypropylene preparedwith pores, that is inert to the reaction of interest and that havepores of size permeable to desired components of the reaction medium.

More than one data storage device may be in proximity to or contact witha matrix particle, or more than one matrix particle may be in contactwith on device. For example, microplates, such as microtiter plates orother such high density format [i.e. 96 or 384 or more wells per plate,such as those available from Nunc, Naperville, Ill., Costar, CambridgeMass., and Millipore, Bedford, Mass.] with the recording devicecontaining the data storage unit [remotely programmable memory] embeddedin each well or vials [typically with a 1 ml or smaller capacity] withan embedded recording device may be manufactured.

In a preferred embodiment, the recording device is a semiconductor thatis approximately 10 mm or less in its largest dimension and the matrixmaterial is a particle, such as a polystyrene bead. The device and aplurality of particles, referred to as “beads”, typically about 1 mg toabout 50 mg, but larger size vessels and amounts up to 1000 mg,preferably 50 to about 200 mg, are sealed in chemically inert poroussupports, such as polypropylene formed so that it has pores of aselected size that excludes the particles but permits passage of theexternal medium. For example, a single device and a plurality ofparticles may be sealed in a porous or semi-permeable inert material toproduce a microvessel [such as the MICROKAN™] such as a teflon orpolypropylene or membrane that is permeable to the components of themedium, or they may be contained in a small closable container that hasat least one dimension that is porous or is a semi-permeable tube.Typically such tube, which preferably has an end that can be opened andsealed or closed tightly. These microvessels preferably have a volume ofabout 200-500 mm³, but can have larger volumes, such as greater than 500mm³ [or 1000 mm³] at least sufficient to contain at least 200 mg ofmatrix particles, such as about 500-3000 mm³, such as 1000-2000 or 1000to 1500, with preferred dimensions of about 1-10 mm in diameter and 5 to20 mm in height, more preferably about 5 mm by 15 mm, or larger, such asabout 1-6 cm by 1-6 cm. The porous wall should be non-collapsible with apore size in the range of 70 μM to about 100 μM, but can be selected tobe semi-permeable for selected components of the medium in which themicrovessel is placed. The preferred geometry of these combinations iscylindrical. These porous microvessels may be sealed by heat or may bedesigned to snap or otherwise close. In some embodiments they aredesigned to be reused. In other embodiments, the microvessel MICROKAN™with closures may be made out of non-porous material, such as a tube inthe conical shape or other geometry.

Also provided herein are tubular devices in which the recording deviseis enclosed in a solid polymer, such as a polypropylene, which is thenradiation grafted with selected monomers to produce a surface suitablefor chemical synthesis and linkage of molecules or biological particles.

Other devices of interest, are polypropylene supports, generally about5-10 mm in the largest dimension, and preferably a cube or other suchshape, that are marked with a code, and tracked using a remote memory.

These microvessels can be marked with a code, such as a bar code,alphanumeric code or other mark, for identification, particularly inembodiments in which the memory is not in proximity to the matrix, butis remote therefrom and used to store information regarding each codedvessel.

The combination of matrix with memory is used by contacting it with,linking it to, or placing it in proximity with a molecule or biologicalparticle, such as a virus or phage particle, a bacterium or a cell, toproduce a second combination of a matrix with memory and a molecule orbiological particle. In certain instances, such combinations of matrixwith memory or combination of matrix with memory and molecule orbiological particle may be prepared when used or may be prepared beforeuse and packaged or stored as such for futures use. The matrix withmemory when linked or proximate to a molecule or biological particle isherein referred to as a microreactor.

The miniature recording device containing the data storage unit(s) withremotely programmable memory, includes, in addition to the remotelyprogrammable memory, means for receiving information for a storage inthe memory and for retrieving information stored in the memory. Suchmeans is typically an antenna, which also serves to provide power in apassive device when combined with a rectifier circuit to convertreceived energy, such as RF, into voltage, that can be tuned to adesired electromagnetic frequency to program the memory. Power foroperation of the recording device may also be provided by a batteryattached directly to the recording device, to create an active device,or by other power sources, including light and chemical reactions,including biological reactions, that generate energy.

Preferred frequencies are any that do not substantially alter themolecular and biological interactions of interest, such as those thatare not substantially absorbed by the molecules or biological particleslinked to the matrix or in proximity of the matrix, and that do notalter the support properties of the matrix. Radio frequencies arepresently preferred, but other frequencies, such as radar, or opticallasers will be used, as long as the selected frequency or optical laserdoes not interfere with the interactions of the molecules or biologicalparticles of interest. Thus, information in the form of data pointscorresponding to such information is stored in and retrieved from thedata storage device by application of a selected electromagneticradiation frequency, which preferably is selected to avoid interferencefrom any background electromagnetic radiation.

The preferred miniature recording device for use in the combinationsherein is a single substrate of a size preferably less than about 10 to20 mm³ [or 10-20 mm in its largest dimension], that includes a remotelyprogrammable data storage unit(s) [memory], preferably a non-volatilememory, and an antenna for receiving or transmitting an electromagneticsignal [and in some embodiments for supplying power in passive deviceswhen combined with a rectifier circuit] preferably a radio frequencysignal; the antenna, rectifier circuit, memory and other components arepreferably integrated onto a single substrate, thereby minimizing thesize of the device. An active device, i.e., one that does not rely onexternal sources for providing voltage for operation of the memory, mayinclude a battery for power, with the battery attached to the substrate,preferably on the surface of the substrate. Vias through the substratecan then provide conduction paths from the battery to the circuitry onthe substrate. The device is rapidly or substantially instantaneouslyprogrammable, preferably in less than 5 seconds, more preferably inabout 1 second, and more preferably in about 50 to 100 milliseconds orless, and most preferably in about 1 millisecond or less. In a passivedevice that relies upon external transmissions to generate sufficientvoltage to operate, write to and read from an electronic recordingdevice, the preferred memory is non-volatile, permanent, and relies onantifuse-based architecture or flash memory. Other memories, such aselectrically programmable erasable read only memories [EEPROMs] basedupon other architectures also can be used in passive devices. In activerecording devices that have batteries to assure continuous poweravailability, a broader range of memory devices may be used in additionto those identified above. These memory devices include dynamic randomaccess memories [DRAMS, which refer to semiconductor volatile memorydevices that allow random input/output of stored information; see, e.g.,U.S. Pat. Nos. 5,453,633, 5,451,896, 5,442,584, 5,442,212 and5,440,511], that permit higher density memories, and EEPROMs.

Containers, such as vials, tubes, microtiter plates, capsules and thelike, which are in contact with a recording device that includes a datastorage unit with programmable memory are also provided. The containeris typically of a size used in immunoassays or hybridization reactions,generally a liter or less, typically less than 100 ml, and often lessthan about 10 ml in volume. Alternatively the container can be in theform of a plurality of wells, such as a microtiter plate, each wellhaving about 1 to 1.5 ml or less in volume. The container istransmissive to the electromagnetic radiation, such as radiofrequencies, infrared wavelengths, radar, ultraviolet wavelengths,microwave frequencies, visible wavelengths, X-rays or laser light, usedto program the recording device.

Methods for electromagnetically tagging molecules or biologicalparticles are provided. Such tagging is effected by placing themolecules or biological particles of interest in proximity with therecording device or with the matrix with memory, and programming orencoding the identity of the molecule or synthetic history of themolecules or batch number or other identifying information into thememory. The, thus identified molecule or biological particle is thenused in the reaction or assay of interest and tracked by virtue of itslinkage to the matrix with memory, its proximity to the matrix withmemory or its having been linked or in proximity to the matrix [i.e.,its association with], which can be queried at will to identify themolecule or biological particle. The tagging and/or reaction or assayprotocols may be automated. Automation will use robotics with integratedmatrix with memory plated based or particulate matrix with memoryautomation [see, U.S. Pat. No. 5,463,564, which provides an automatediterative method of drug design].

In particular, methods for tagging constituent members of combinatoriallibraries and other libraries or mixtures of diverse molecules andbiological particles are provided. These methods involveelectromagnetically tagging molecules, particularly constituent membersof a library, by contacting the molecules or biological particles orbringing such molecules or particles into proximity with a matrix withmemory and programming the memory with retrievable information fromwhich the identity, synthesis history, batch number or other identifyinginformation can be retrieved. The contact is preferably effected bycoating, completely or in part, the recording device with memory withthe matrix and then linking, directly or via linkers, the molecule orbiological particle of interest to the matrix support. The memories canbe coated with a protective coating, such as a glass or silicon, whichcan be readily derivatized for chemical linkage or coupling to thematrix material. In other embodiments, the memories can be coated withmatrix, such as for example dipping the memory into the polymer prior topolymerization, and allowing the polymer to polymerize on the surface ofthe memory.

If the matrices are used for the synthesis of the constituent molecules,the memory of each particle is addressed and the identity of the addedcomponent is encoded in the memory at [before, during, or preferablyafter] each step in the synthesis. At the end of the synthesis, thememory contains a retrievable record of all of the constituents of theresulting molecule, which can then be used, either linked to thesupport, or following cleavage from the support in an assay or forscreening or other such application. If the molecule is cleaved from thesupport with memory, the memory must remain in proximity to the moleculeor must in some manner be traceable [i.e., associated with] to themolecule. Such synthetic steps may be automated.

In preferred embodiments, the matrix with memory with linked molecules[or biological particles] are mixed and reacted with a sample accordingto a screening or assay protocol, and those that react are isolated. Theidentity of reacted molecules can then be ascertained by remotelyretrieving the information stored in the memory and decoding it toidentify the linked molecules.

Compositions containing combinations of matrices with memories andcompositions of matrices with memories and molecules or biologicalparticles are also provided. In particular, coded or electronicallytagged libraries of oligonucleotides, peptides, proteins, non-peptideorganic molecules, phage display, viruses and cells are provided.Particulate matrices, such as polystyrene beads, with attached memories,and continuous matrices, such as microtiter plates or slabs or polymer,with a plurality of embedded or attached memories are provided.

These combinations of matrix materials with memories and combinations ofmatrices with memories and molecules or biological particles may be usedin any application in which support-bound molecules or biologicalparticles are used. Such applications include, but are not limited todiagnostics, such as immunoassays, drug screening assays, combinatorialchemistry protocols and other such uses. These matrices with memoriescan be used to tag cells for uses in cell sorting, to identify moleculesin combinatorial syntheses, to label monoclonal antibodies, to tagconstituent members of phage displays, affinity separation procedures,to label DNA and RNA, in nucleic acid amplification reactions [see,e.g., U.S. Pat. No. 5,403,484; U.S. Pat. No. 5,386,024; U.S. Pat. No.4,683,202 and, for example International PCT Application WO/94 02634,which describes the use of solid supports in connection with nucleicacid amplification methods], to label known compounds, particularlymixtures of known compounds in multianalyte analyses], to therebyidentify unknown compounds, or to label or track unknowns and therebyidentify the unknown by virtue of reaction with a known. Thus, thematrices with memories are particularly suited for high throughputscreening applications and for multianalyte analyses.

Systems and methods for recording and reading or retrieving theinformation in the data storage devices regarding the identity orsynthesis of the molecules or biological particles are also provided.The systems for recording and reading data include: a host computer orother encoder/decoder instrument having a memory for storing datarelating to the identity or synthesis of the molecules, and atransmitter means for receiving a data signal and generating a signalfor transmitting a data signal; and a recording device that includes aremotely programmable, preferably non-volatile, memory and transmittermeans for receiving a data signal and generating at least a transmittedsignal and for providing a write signal to the memory in the recordingdevice. The host computer stores transmitted signals from the memorieswith matrices, and decodes the transmitted information.

In particular, the systems include means for writing to and reading fromthe memory device to store and identify each of the indicators thatidentify or track the molecules and biological particles. The systemsadditionally include the matrix material in physical contact with orproximate to the recording device, and may also include a device forseparating matrix particles with memory so that each particle or memorycan be separately programmed.

Methods for tagging molecules and biological particles by contacting,either directly or indirectly, a molecule or biological particle with arecording device; transmitting from a host computer or decoder/encoderinstrument to the device electromagnetic radiation representative of adata signal corresponding to an indicator that either specifies one of aseries of synthetic steps or the identity or other information foridentification of the molecule or biological particle, whereby the datapoint representing the indicator is written into the memory, areprovided.

Methods for reading identifying information from recording deviceslinked to or in contact with or in proximity to or that had been incontact with or proximity to a electromagnetically tagged molecule orelectromagnetically tagged biological particles are provided. Thesemethods include the step of exposing the recording device containing thememory in which the data are stored to electromagnetic radiation [EM];and transmitting to a host computer or decoder/encoder instrument anindicator representative of a the identity of a molecule or biologicalparticle or identification of the molecule or biological particle linkedto, in proximity to or associated with the recording device.

One, two, three and N-dimensional arrays of the matrices with memoriesare also provided. Each memory is programmed with its position in thearray. Such arrays may be used for blotting, if each matrix particle iscoated on one at least one side with a suitable material, such asnitrocellulose. For blotting, each memory is coated on at least one sidewith the matrix material and arranged contiguously to adjacent memoriesto form a substantially continuous sheet. After blotting, the matrixparticles may be separated and reacted with the analyte of interest[e.g., a labeled antibody or oligonucleotide or other ligand], afterwhich the physical position of the matrices to which analyte binds maybe determined. The amount of bound analyte, as well as the kinetics ofthe binding reaction, may also be quantified. Southern, Northern,Western, dot blot and other such assays using such arrays are provided.Dimensions beyond three can refer to additional unique identifyingparameters, such as batch number, and simultaneous analysis of multipleblots.

Assays that use combinations of (i) a miniature recording device thatcontains one or more programmable data storage devices [memories] thatcan be remotely programmed and read; and (ii) a matrix, such as aparticulate support used in chemical syntheses, are provided. The remoteprogramming and reading is preferably effected using electromagneticradiation.

Also provided are scintillation proximity assays, HTRF, FP, FET and FRETassays in which the memories are in proximity with or are in physicalcontact with the matrix that contains scintillant for detectingproximate radionucleotide signals or fluorescence. In addition,embodiments that include a memory device that also detects occurrence ofa reaction are provided.

Molecular libraries, DNA libraries, peptide libraries, biologicalparticle libraries, such as phage display libraries, in which theconstituent molecules or biological particles are combined with a solidsupport matrix that is combined with a data storage unit with aprogrammable memory are provided.

Affinity purification protocols in which the affinity resin is combinedwith a recording device containing a data storage unit with aprogrammable memory are also provided.

Immunological, biochemical, cell biological, molecular biological,microbiological, and chemical assays in which memory with matrixcombinations are used are provided. For example immunoassays, such asenzyme linked immunosorbent assays [ELISAs] in which at least oneanalyte is linked to a solid support matrix that is combined with arecording device containing a data storage unit with a programmable,preferably remotely programmable and non-volatile, memory are provided.

Of particular interest herein, are multiprotocol applications [such asmultiplexed assays or coupled synthetic and assay protocols] in whichthe matrices with memories are used in a series [more than one] ofreactions, a series [more than one] of assays, and/or a series of moreor more reactions and one or more assays, typically on a single platformor coupled via automated analysis instrumentation. As a result synthesisis coupled to screening.

DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts combinatorial synthesis of chemical libraries on matrixsupports with memories. A, B, C . . . represent the chemical buildingblocks; a, b, c . . . represent the codes stored in memory thatcorrespond to each of A, B, C, . . . , respectively. S_(a), S_(b), S_(c). . . represent respective signals sent to memory.

FIG. 2 depicts combinatorial synthesis of peptides on a matrix withmemory. Each amino acid has a corresponding code, a, b, c . . . , in thematrix memory, and L represents a Linker between the memory device andthe pharmacophore.

FIG. 3 depicts combinatorial synthesis of oligonucleotides on matrixsupports with memories. A, G, T and C represent nucleotides, and a, g,t, and c represent the electronic codes stored in memory that correspondto each of A, G T and C, respectively. The phosphoramidite method ofoligonucleotide synthesis is performed by methods known to those ofskill in the art [see, e.g., Brown et al. (1991) “Modern machine-aidedmethods of oligodeoxyribonucleotide synthesis” in OligonucleotidesAnalogues EDITOR: Eckstein, Fritz (Ed), IRL, Oxford, UK., pp. 1-24, esp.pp. 4-7].

FIG. 4 depicts generation of a chemical library, such as a library oforganic molecules, in which R₁, R₂, R₃ are substituents on selectedmolecule, such as a pharmacophore monomer, each identified with adifferent signal, depicted as 1, 2, or 3, from the classes S₁, S₂, S₃,respectively. The circle represents an organic pharmacophore. If R₁-R₃are the same, and selected from among the same 50 choices, then thecomplete library contains 50³=125,000 members. If R₁-R₃ selected fromamong different sets of choices, then the resulting library hascorrespondingly more members. Each matrix memory can be encoded withinformation that represents the R_(n), added and class [S_(n)] therebyproviding a unique code for each library member.

FIG. 5 is a block diagram of the data storage means and supportingelectrical components of a preferred embodiment.

FIG. 6 is a diagrammatic view of the memory array within the recordingdevice, and the corresponding data stored in the host computer memory.

FIG. 7 is an illustration of an exemplary apparatus for separating thematrix particles with memories for individual exposure to an EM signal.

FIG. 8 is an illustration of a second exemplary embodiment of anapparatus for separating matrix particles for individual exposure to anoptical signal.

FIG. 9 is a diagrammatic view of the memory array within the recordingdevice, the corresponding data stored in the host computer memory, andincluded photodetector with amplifier and gating transistor.

FIG. 10 is a scheme for the synthesis of the 8 member RF encodedcombinatorial decameric peptide library described in EXAMPLE 4. Allcouplings were carried out in DMF at ambient temperature for 1 h [twocouplings per amino acid], using PyBOP and EDIA or DIEA. Deprotectionconditions: 20% piperidine in DMF, ambient temperature, 30 min; Cleavageconditions: 1,2-ethanedithiol:thioanisole:water:phenol:trifluoroaceticacid [1.5:3:3:4.5:88, w/w], ambient temperature, 1.5 h.

FIG. 11 is a side elevation of a preferred embodiment of a microvessel.

FIG. 12 is a sectional view, with portions cut away, taken along line12-12 of FIG. 11.

FIG. 13 is a sectional view taken along line 13-13 of FIG. 12.

FIG. 14 is a perspective view of an alternative embodiment of amicrovessel, with the end cap separated.

FIG. 15 is a side elevation view of the microvessel of FIG. 14, with aportion cut away.

FIG. 16 is a sectional view taken along line 16-16 of FIG. 15.

FIG. 17 is a perspective view of an exemplary write/read station.

FIG. 18 is a flow diagram of the operation of the system of FIG. 17.

FIG. 19 Fluorescent solid supports: application in solid phase synthesisof direct SPA.

FIG. 20 Coded macro “beads” for efficient combinatorial synthesis.

FIG. 21 Preparation and use of tubular microvessel in which thecontainer is radiation grafted with monomers for use as a supportmatrix.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as is commonly understood by one of skill in theart to which this invention belongs. All patents and publicationsreferred to herein are, unless noted otherwise, incorporated byreference in their entirety.

As used herein, a matrix refers to any solid or semisolid or insolublesupport to which the memory device and/or the molecule of interest,typically a biological molecule, organic molecule or biospecific ligandis linked or contacted. Typically a matrix is a substrate materialhaving a rigid or semi-rigid surface. In many embodiments, at least onesurface of the substrate will be substantially flat, although in someembodiments it may be desirable to physically separate synthesis regionsfor different polymers with, for example, wells, raised regions, etchedtrenches, or other such topology. Matrix materials include any materialsthat are used as affinity matrices or supports for chemical andbiological molecule syntheses and analyses, such as, but are not limitedto: polystyrene, polycarbonate, polypropylene, nylon, glass, dextran,chitin, sand, pumice, teflon, agarose, polysaccharides, dendrimers,buckyballs, polyacrylamide, Kieselguhr-polyacrlamide non-covalentcomposite, polystyrene-polyacrylamide covalent composite,polystyrene-PEG [polyethyleneglycol] composite, silicon, rubber, andother materials used as supports for solid phase syntheses, affinityseparations and purifications, hybridization reactions, immunoassays andother such applications. The matrix herein may be particulate or may bein the form of a continuous surface, such as a microtiter dish or well,a glass slide, a silicon chip, a nitrocellulose sheet, nylon mesh, orother such materials. When particulate, typically the particles have atleast one dimension in the 5-10 mm range or smaller. Such particles,referred collectively herein as “beads”, are often, but not necessarily,spherical. Such reference, however, does not constrain the geometry ofthe matrix, which may be any shape, including random shapes, needles,fibers, elongated, etc. The “beads” may include additional components,such as magnetic or paramagnetic particles [see, e.g., Dyna beads(Dynal, Oslo, Norway)] for separation using magnets, fluophores andother scintillants, as long as the additional components do notinterfere with chemical reactions, data entry or retrieval from thememory.

As used herein, scintillants include, 2,5-diphenyloxazole [PPO],anthracene, 2-(4′-tert-butylphenyl)-5-(4″-biphenyl)-1,3,4-oxadiazole[butyl-PBD]; 1-phenyl-3-mesityl-2-pyrazoline [PMP], with or withoutfrequency shifters, such as 1,4,-bis[5-phenyl(oxazolyl)benzene][POPOP];p-bis-o-methylstyrylbenzene [bis-MSB]. Combinations of these fluors,such as PPO and POPOP or PPO and bis-MSB, in suitable solvents, such asbenzyltoluene [see, e.g., U.S. Pat. No. 5,410,155], are referred to asscintillation cocktails.

As used herein a luminescent moiety refers to a scintillant or fluophorused in scintillation proximity assays or in non-radioactive energytransfer assays, such as HTRF assays.

As used herein, matrix particles refer to matrix materials that are inthe form of discrete particles. The particles have any shape anddimensions, but typically have at least one dimension that is 100 mm orless, preferably 50 mm or less, more preferably 10 mm or less, andtypically have a size that is 100 mm³ or less, preferably 50 mm³ orless, more preferably 10 mm³ or less, and most preferably 1 mm³ or less.The matrices may also be continuous surfaces, such as microtiter plates[e.g., plates made from polystyrene or polycarbonate or derivativesthereof commercially available from Perkin Elmer Cetus and numerousother sources, and Covalink trays [Nunc], microtiter plate lids or atest tube, such as a 1 ml Eppendorf tube. Matrices that are in the formof containers refers to containers, such as test tubes and microplatesand vials that are typically used for solid phase syntheses ofcombinatorial libraries or as pouches, vessels, bags, and microvesselsfor screening and diagnostic assays. Thus, a container used for chemicalsyntheses refers to a container that typically has a volume of about 1liter, generally 100 ml, and more often 10 ml or less, 5 ml or less,preferably 1 ml or less, and as small as about 50 μl-500 μl, such as 100μl or 250 μl. This also refers to multi-well plates, such as microtiterplates [96 well, 384 well or other density format]. Such microtiterplate will typically contain a recording device in, on, or otherwise incontact with in each of a plurality of wells.

As used herein, a matrix with a memory refers to a combination of amatrix with a miniature recording device that stores multiple bits ofdata by which the matrix may be identified, preferably in a non-volatilememory that can be written to and read from by transmission ofelectromagnetic radiation from a remote host, such as a computer. Byminiature is meant of a size less than about 10-20 mm³ [or 10-20 mm inthe largest dimension]. Preferred memory devices or data storage unitsare miniature and are preferably smaller than 10-20 mm³ [or 10-20 mm inits largest dimension] dimension, more preferably less than 5 mm³, mostpreferably about 1 mm³ or smaller.

As used herein, a microreactor refers to combinations of matrices withmemories with associated, such as linked or proximate, biologicalparticles or molecules. It is produced, for example, when the moleculeis linked thereto or synthesized thereon. It is then used in subsequentprotocols, such as immunoassays and scintillation proximity assays.

As used herein, a combination herein called a microvessel [e.g., anMICROKAN™] refers to a combination in which a single device [or morethan one device] and a plurality of particles are sealed in a porous orsemi-permeable inert material, such as teflon or polypropylene ormembrane that is permeable to the components of the medium, but retainsthe particles and memory, or are sealed in a small closable containerthat has at least one dimension that is porous or semi-permeable.Typically such microvessels, which preferably have at least one end thatcan be opened and sealed or closed tightly, has a volume of about200-500 mm³, with preferred dimensions of about 1-10 mm in diameter and5 to 20 mm in height, more preferably about 5 mm by 15 mm. The porouswall should be non-collapsible with a pore size in the range of 70 μM toabout 100 μM, but can be selected to be semi-permeable for selectedcomponents of the reaction medium.

As used herein, a memory is a data storage unit [or medium] withprogrammable memory, preferably a non-volatile memory.

As used herein, programming refers to the process by which data orinformation is entered and stored in a memory. A memory that isprogrammed is a memory that contains retrievable information.

As used herein, remotely programmable, means that the memory can beprogrammed without direct physical or electrical contact or can beprogrammed from a distance, typically at least about 10 mm, althoughshorter distances may also be used, such as instances in which theinformation comes from surface or proximal reactions or from an adjacentmemory or in instances, such as embodiments in which the memories arevery close to each other, as in microtiter plate wells or in an array.

As used herein, a recording device [or memory device] is an apparatusthat includes the data storage unit with programmable memory, and, ifnecessary, means for receiving information and for transmittinginformation that has been recorded. It includes any means needed or usedfor writing to and reading from the memory. The recording devicesintended for use herein, are miniature devices that preferably aresmaller than 10-20 mm³ [or 10-20 mm in their largest dimension], andmore preferably are closer in size to 1 mm³ or smaller that contain atleast one such memory and means for receiving and transmitting data toand from the memory.

As used herein, a data storage unit with programmable memory includesany data storage means having the ability to record multiple discretebits of data, which discrete bits of data may be individually accessed[read] after one or more recording operations. Thus, a matrix withmemory is a combination of a matrix material with a miniature datastorage unit.

As used herein, programmable means capable of storing unique datapoints. Addressable means having unique locations that may be selectedfor storing the unique data points.

As used herein, reaction verifying and reaction detecting areinterchangeable and refer to the combination that also includes elementsthat detect occurrence of a reaction or event of interest between theassociated molecule or biological particle and its environment [i.e.,detects occurrence of a reaction, such as ligand binding, by virtue ofemission of EM upon reaction or a change in pH or temperature or otherparameter].

As used herein, a host computer or decoder/encoder instrument is aninstrument that has been programmed with or includes information [i.e.,a key] specifying the code used to encode the memory devices. Thisinstrument or one linked thereto transmits the information and signalsto the recording device and it, or another instrument, receives theinformation transmitted from the recording device upon receipt of theappropriate signal. This instrument thus creates the appropriate signalto transmit to the recording device and can interpret transmittedsignals. For example, if a “1” is stored at position 1,1 in the memoryof the recording device means, upon receipt of this information, thisinstrument or computer can determine that this means the linked moleculeis, for example, a peptide containing alanine at the N-terminus, anorganic group, organic molecule, oligonucleotide, or whatever thisinformation has been predetermined to mean. Alternatively, theinformation sent to and transmitted from the recording device can beencoded into the appropriate form by a person.

As used herein, an electromagnetic tag is a recording device that has amemory that contains unique data points that correspond to informationthat identifies molecules or biological particles linked to, directly orindirectly, in physical contact with or in proximity [or associatedwith] to the device. Thus, electromagnetic tagging is the process bywhich identifying or tracking information is transmitted [by any meansand to any recording device memory, including optical and magneticstorage media] to the recording device.

As used herein, proximity means within a very short distance, generallyless than 0.5 inch, typically less than 0.2 inches. In particular,stating that the matrix material and memory, or the biological particleor molecule and matrix with memory are in proximity means that, they areat least or at least were in the same reaction vessel or, if the memoryis removed from the reaction vessel, the identity of the vesselcontaining the molecules or biological particles with which the memorywas proximate or linked is tracked or otherwise known.

As used herein, associated with means that the memory must remain inproximity to the molecule or biological particle or must in some mannerbe traceable to the molecule or biological particle. For example, if amolecule is cleaved from the support with memory, the memory must insome manner be identified as having been linked to the cleaved molecule.Thus, a molecule or biological particle that had been linked to or inproximity to a matrix with memory is associated with the matrix ormemory if it can be identified by querying the memory.

As used herein, antifuse refers to an electrical device that isinitially an open circuit that becomes a closed circuit duringprogramming, thereby providing for non-volatile memory means and, whenaccompanied by appropriate transceiver and rectification circuitry,permitting remote programming and, hence identification. In practice, anantifuse is a substantially nonconductive structure that is capable ofbecoming substantially conductive upon application of a predeterminedvoltage, which exceeds a threshold voltage. An antifuse memory does notrequire a constant voltage source for refreshing the memory and,therefore, may be incorporated in a passive device. Other memories thatmay be used include, but are not limited to: EEPROMS, DRAMS and flashmemories.

As used herein, flash memory is memory that retains information whenpower is removed [see, e.g., U.S. Pat. No. 5,452,311, U.S. Pat. No.5,452,251 and U.S. Pat. No. 5,449,941]. Flash memory can be rewritten byelectrically and collectively erasing the stored data, and then byprogramming.

As used herein, passive device refers to an electrical device which doesnot have its own voltage source and relies upon a transmitted signal toprovide voltage for operation.

As used herein, electromagnetic [EM] radiation refers to radiationunderstood by skilled artisans to be EM radiation and includes, but isnot limited to radio frequency [RF], infrared [IR], visible, ultraviolet[UV], radiation, sonic waves, X-rays, and laser light.

As used herein, information identifying or tracking a biologicalparticle or molecule, refers to any information that identifies themolecule or biological particle, such as, but not limited to theidentity particle [i.e. its chemical formula or name], its sequence, itstype, its class, its purity, its properties, such as its bindingaffinity for a particular ligand. Tracking means the ability to follow amolecule or biological particle through synthesis and/or process steps.The memory devices herein store unique indicators that represent any ofthis information.

As used herein, combinatorial chemistry is a synthetic strategy thatproduces diverse, usually large, chemical libraries. It is thesystematic and repetitive, covalent connection of a set, the basis set,of different monomeric building blocks of varying structure to eachother to produce an array of diverse molecules [see, e.g., Gallop et al.(1994) J. Medicinal Chemistry 37:1233-1251]. It also encompasses otherchemical modifications, such as cyclizations, eliminations, cleavages,etc., that are carried in manner that generates permutations and therebycollections of diverse molecules.

As used herein, a biological particle refers to a virus, such as a viralvector or viral capsid with or without packaged nucleic acid, phage,including a phage vector or phage capsid, with or without encapsulatednucleotide acid, a single cell, including eukaryotic and prokaryoticcells or fragments thereof, a liposome or micellar agent or otherpackaging particle, and other such biological materials.

As used herein, the molecules in the combinations include any molecule,including nucleic acids, amino acids, other biopolymers, and otherorganic molecules, including peptidomimetics and monomers or polymers ofsmall organic molecular constituents of non-peptidic libraries, that maybe identified by the methods here and/or synthesized on matrices withmemories as described herein.

As used herein, the term “bio-oligomer” refers to a biopolymer of lessthan about 100 subunits. A bio-oligomer includes, but is not limited to,a peptide, i.e., containing amino acid subunits, an oligonucleotide,i.e., containing nucleoside subunits, a peptide-oligonucleotide chimera,peptidomimetic, and a polysaccharide.

As used herein, the term “sequences of random monomer subunits” refersto polymers or oligomers containing sequences of monomers in which anymonomer subunit may precede or follow any other monomer subunit.

As used herein, the term “library” refers to a collection ofsubstantially random compounds or biological particles expressing randompeptides or proteins or to a collection of diverse compounds. Ofparticular interest are bio-oligomers, biopolymers, or diverse organiccompounds or a set of compounds prepared from monomers based on aselected pharmacophore.

As used herein, an analyte is any substance that is analyzed or assayedin the reaction of interest. Thus, analytes include the substrates,products and intermediates in the reaction, as well as the enzymes andcofactors.

As used herein, multianalyte analysis is the ability to measure manyanalytes in a single specimen or to perform multiple tests from a singlespecimen. The methods and combinations herein provide means to identifyor track individual analytes from among a mixture of such analytes.

As used herein, a fluophore or a fluor is a molecule that readilyfluoresces; it is a molecule that emits light following interaction withradiation. The process of fluorescence refers to emission of a photon bya molecule in an excited singlet state. For scintillation assays,combinations of fluors are typically used. A primary fluor that emitslight following interaction with radiation and a secondary fluor thatshifts the wavelength emitted by the primary fluor to a higher moreefficiently detected wavelength.

As used herein, a peptidomimetic is a compound that mimics theconformation and certain stereochemical features of the biologicallyactive form of a particular peptide. In general, peptidomimetics aredesigned to mimic certain desirable properties of a compound but not theundesirable features, such as flexibility leading to a loss of thebiologically active conformation and bond breakdown. For example,methylenethio bioisostere [CH₂S] has been used as an amide replacementin enkephalin analogs [see, e.g., Spatola, A. F. Chemistry andBiochemistry of Amino Acids, Peptides, and Proteins [Weinstein, B, Ed.,Vol. 7, pp. 267-357, Marcel Dekker, New York (1983); and Szelke et al.(1983) In Peptides: Structure and Function, Proceedings of the EighthAmerican Peptide Symposium, Hruby and Rich, Eds., pp. 579-582, PierceChemical Co., Rockford, Ill.].

As used herein, complete coupling means that the coupling reaction isdriven substantially to completion despite or regardless of thedifferences in the coupling rates of individual components of thereaction, such as amino acids In addition, the amino acids, or whateveris being coupled, are coupled to substantially all available couplingsites on the solid phase support so that each solid phase support willcontain essentially only one species of peptide.

As used herein, the biological activity or bioactivity of a particularcompound includes any activity induced, potentiated or influenced by thecompound In vivo or in vitro. It also includes the abilities, such asthe ability of certain molecules to bind to particular receptors and toinduce [or modulate] a functional response. It may be assessed by invivo assays or by in vitro assays, such as those exemplified herein.

As used herein, pharmaceutically acceptable salts, esters or otherderivatives of the compounds include any salts, esters or derivativesthat may be readily prepared by those of skill in this art using knownmethods for such derivatization and that produce compounds that may beadministered to animals or humans without substantial toxic effects andthat either are pharmaceutically active or are prodrugs. For example,hydroxy groups can be esterified or etherified.

As used herein, substantially pure means sufficiently homogeneous toappear free of readily detectable impurities as determined by standardmethods of analysis, such as thin layer chromatography [TLC], massspectrometry [MS], size exclusion chromatography, gel electrophoresis,particularly agarose and polyacrylamide gel electrophoresis [PAGE] andhigh performance liquid chromatography [HPLC], used by those of skill inthe art to assess such purity, or sufficiently pure such that furtherpurification would not detectably alter the physical and chemicalproperties, such as enzymatic and biological activities, of thesubstance. Methods for purification of the compounds to producesubstantially chemically pure compounds are known to those of skill inthe art. A substantially chemically pure compound may, however, be amixture of stereoisomers. In such instances, further purification mightincrease the specific activity of the compound.

As used herein, adequately pure or “pure” per se means sufficiently purefor the intended use of the adequately pure compound.

As used herein, biological activity refers to the in vivo activities ofa compound or physiological responses that result upon in vivoadministration of a compound, composition or other mixture. Biologicalactivity, thus, encompasses therapeutic effects and pharmaceuticalactivity of such compounds, compositions and mixtures.

As used herein, a prodrug is a compound that, upon in vivoadministration, is metabolized or otherwise converted to thebiologically, pharmaceutically or therapeutically active form of thecompound. To produce a prodrug, the pharmaceutically active compound ismodified such that the active compound will be regenerated by metabolicprocesses. The prodrug may be designed to alter the metabolic stabilityor the transport characteristics of a drug, to mask side effects ortoxicity, to improve the flavor of a drug or to alter othercharacteristics or properties of a drug. By virtue of knowledge ofpharmacodynamic processes and drug metabolism in vivo, those of skill inthis art, once a pharmaceutically active compound is known, can designprodrugs of the compound [see, e.g., Nogrady (1985) Medicinal ChemistryA Biochemical Approach, Oxford University Press, New York, pages388-392].

As used herein, amino acids refer to the naturally-occurring amino acidsand any other non-naturally occurring amino acids, and also thecorresponding D-isomers. It is also understood that certain amino acidsmay be replaced by substantially equivalent non-naturally occurringvariants thereof, such as D-Nva, D-Nle, D-Alle, and others listed withthe abbreviations below or known to those of skill in this art.

As used herein, hydrophobic amino acids include Ala, Val, Leu, Ile, Pro,Phe, Trp, and Met, the non-naturally occurring amino acids and thecorresponding D isomers of the hydrophobic amino acids, that havesimilar hydrophobic properties; the polar amino acids include Gly, Ser,Thr, Cys, Tyr, Asn, Gln, the non-naturally occurring amino acids and thecorresponding D isomers of the polar amino acids, that have similarproperties, the charged amino acids include Asp, Glu, Lys, Arg, His, thenon-naturally occurring amino acids and the corresponding D isomers ofthese amino acids.

As used herein, Southern, Northern, Western and dot blot proceduresrefer to those in which DNA, RNA and protein patterns, respectively, aretransferred for example, from agarose gels, polyacrylamide gels or othersuitable medium that constricts convective motion of molecules, tonitrocellulose membranes or other suitable medium for hybridization orantibody or antigen binding are well known to those of skill in this art[see, e.g., Southern (1975) J. Mol. Biol. 98:503-517; Ketner et al.(1976) Proc. Natl. Acad. Sci. U.S.A. 73:1102-1106; Towbin et al. (1979)Proc. Natl. Acad. Sci. U.S.A. 76:4350].

As used herein, a receptor refers to a molecule that has an affinity fora given ligand. Receptors may be naturally-occurring or syntheticmolecules. Receptors may also be referred to in the art as anti-ligands.As used herein, both terms, receptor and anti-ligand areinterchangeable. Receptors can be used in their unaltered state or asaggregates with other species. Receptors may be attached, covalently ornoncovalently, or in physical contact with, to a binding member, eitherdirectly or indirectly via a specific binding substance or linker.Examples of receptors, include, but are not limited to: antibodies, cellmembrane receptors surface receptors and internalizing receptors,monoclonal antibodies and antisera reactive with specific antigenicdeterminants [such as on viruses, cells, or other materials], drugs,polynucleotides, nucleic acids, peptides, cofactors, lectins, sugars,polysaccharides, cells, cellular membranes, and organelles.

Examples of receptors and applications using such receptors, include butare not restricted to:

a) enzymes: specific transport proteins or enzymes essential to survivalof microorganisms, which could serve as targets for antibiotic [ligand]selection;

b) antibodies: identification of a ligand-binding site on the antibodymolecule that combines with the epitope of an antigen of interest may beinvestigated; determination of a sequence that mimics an antigenicepitope may lead to the development of vaccines of which the immunogenis based on one or more of such sequences or lead to the development ofrelated diagnostic agents or compounds useful in therapeutic treatmentssuch as for auto-immune diseases

c) nucleic acids: identification of ligand, such as protein or RNA,binding sites;

d) catalytic polypeptides: polymers, preferably polypeptides, that arecapable of promoting a chemical reaction involving the conversion of oneor more reactants to one or more products; such polypeptides generallyinclude a binding site specific for at least one reactant or reactionintermediate and an active functionality proximate to the binding site,in which the functionality is capable of chemically modifying the boundreactant [see, e.g., U.S. Pat. No. 5,215,899];

e) hormone receptors: determination of the ligands that bind with highaffinity to a receptor is useful in the development of hormonereplacement therapies; for example, identification of ligands that bindto such receptors may lead to the development of drugs to control bloodpressure; and

f) opiate receptors: determination of ligands that bind to the opiatereceptors in the brain is useful in the development of less-addictivereplacements for morphine and related drugs.

As used herein, antibody includes antibody fragments, such as Fabfragments, which are composed of a light chain and the variable regionof a heavy chain.

As used herein, complementary refers to the topological compatibility ormatching together of interacting surfaces of a ligand molecule and itsreceptor. Thus, the receptor and its ligand can be described ascomplementary, and furthermore, the contact surface characteristics arecomplementary to each other.

As used herein, a ligand-receptor pair or complex formed when twomacromolecules have combined through molecular recognition to form acomplex.

As used herein, an epitope refers to a portion of an antigen moleculethat is delineated by the area of interaction with the subclass ofreceptors known as antibodies.

As used herein, a ligand is a molecule that is specifically recognizedby a particular receptor. Examples of ligands, include, but are notlimited to, agonists and antagonists for cell membrane receptors, toxinsand venoms, viral epitopes, hormones [e.g., steroids], hormonereceptors, opiates, peptides, enzymes, enzyme substrates, cofactors,drugs, lectins, sugars, oligonucleotides, nucleic acids,oligosaccharides, proteins, and monoclonal antibodies.

As used herein, a sensor is a device or apparatus that monitors externalparameters (i.e., conditions), such as ion concentrations, pH,temperatures. Biosensors are sensors that detect biological species.Sensors encompass devices that rely on electrochemical, optical,biological and other such means to monitor the environment.

As used herein, multiplexing refers to performing a series of syntheticand processing steps and/or assaying steps on the same platform [i.e.solid support or matrix] or coupled together as part of the sameautomated coupled protocol, including one or more of the following,synthesis, preferably accompanied by writing to the linked memories toidentify linked compounds, screening, including using protocols withmatrices with memories, and compound identification by querying thememories of matrices associated with the selected compounds. Thus, theplatform refers system in which all manipulations are performed. Ingeneral it means that several protocols are coupled and performedsequentially or simultaneously.

As used herein, a platform refers to the instrumentation or devices inwhich on which a reaction or series of reactions is(are) performed.

As used herein a protecting group refers to a material that ischemically bound to a monomer unit that may be removed upon selectiveexposure to an activator such as electromagnetic radiation and,especially ultraviolet and visible light, or that may be selectivelycleaved. Examples of protecting groups include, but are not limited to:those containing nitropiperonyl, pyrenylmethoxy-carbonyl, nitroveratryl,nitrobenzyl, dimethyl dimethoxybenzyl, 5-bromo-7-nitroindolinyl,o-hydroxy-alpha-methyl cinnamoyl, and 2-oxymethylene anthraquinone.

Also protected amino acids are readily available to those of skill inthis art. For example, Fmoc and Boc protected amino acids can beobtained from Fluka, Bachem, Advanced Chemtech, Sigma, CambridgeResearch Biochemical, Bachem, or Peninsula Labs or other chemicalcompanies familiar to those who practice this art.

As used herein, the abbreviations for amino acids and protective groupsare in accord with their common usage and the IUPAC-IUB Commission onBiochemical Nomenclature [see, (1972) Biochem. 11: 942-944]. Eachnaturally occurring L-amino acid is identified by the standard threeletter code or the standard three letter code with or without the prefix“L-”; the prefix “D-” indicates that the stereoisomeric form of theamino acid is D. For example, as used herein, Fmoc is9-fluorenylmethoxycarbonyl; BOP isbenzotriazol-1-yloxytris(dimethylamino) phosphonium hexafluorophosphate,DCC is dicyclohexylcarbodiimide; DDZ is dimethoxydimethylbenzyloxy; DMTis dimethoxytrityl; FMOC is fluorenylmethyloxycarbonyl; HBTU is2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium; hexafluorophosphateNV is nitroveratryl; NVOC is 6-nitroveratryloxycarbonyl and otherphotoremovable groups; TFA is trifluoroacetic acid; DMF forN,N-dimethylformamide; Boc is tertbutoxycarbonyl; TFA fortrifluoroacetic acid; HF for hydrogen fluoride; HFIP forhexafluoroisopropanol; HPLC for high performance liquid chromatography;FAB-MS for fast atom bombardment mass spectrometry; DCM isdichloromethane, Bom is benzyloxymethyl; Pd/C is palladium catalyst onactivated charcoal; DIC is diisopropylcarbodiimide; DCC isN,N′-dicyclohexylcarbodiimide; [For] is formyl; PyBop isbenzotriazol-1-yl-oxy-trispyrrolidino-phosphonium hexafluorophosphate;POPOP is 1,4,-bis[5-phenyl(oxazolyl)benzene]; PPO is2,5-diphenyloxazole; butyl-PBD is[2-(4′-tert-butylphenyl)-5-(4″-biphenyl)-1,3,4-oxadiazole]; PMP is(1-phenyl-3-mesityl-2-pyrazoline) DIEA is diisopropylethylamine; EDIA isethyldiisopropylethylamine; NMP is N-methylpyrrolidone; NV isnitroveratryl PAL is pyridylalanine; HATU isO(7-azabenzotriaol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate;TFA is trifluoracetic acid, THF is tetrahydrofuran; and EDT is1,2-ethanedithiol.

A. Matrices

Matrices, which are generally insoluble materials used to immobilizeligands and other molecules, have application in many chemical synthesesand separations. Matrices are used in affinity chromatography, in theimmobilization of biologically active materials, and during chemicalsyntheses of biomolecules, including proteins, amino acids and otherorganic molecules and polymers. The preparation of and use of matricesis well known to those of skill in this art; there are many suchmaterials and preparations thereof known. For example,naturally-occurring matrix materials, such as agarose and cellulose, maybe isolated from their respective sources, and processed according toknown protocols, and synthetic materials may be prepared in accord withknown protocols.

Matrices include any material that can act as a support matrix forattachment of the molecules or biological particles of interest and canbe in contact with or proximity to or associated with, preferablyencasing or coating, the data storage device with programmable memory.Any matrix composed of material that is compatible with and upon or inwhich chemical syntheses are performed, including biocompatiblepolymers, is suitable for use herein. The matrix material should beselected so that it does not interfere with the chemistry or biologicalreaction of interest during the time which the molecule or particle islinked to, or in proximity therewith [see, e.g., U.S. Pat. No.4,006,403]. These matrices, thus include any material to which the datastorage device with memory can be attached, placed in proximity thereof,impregnated, encased or otherwise connected, linked or physicallycontacted. Such materials are known to those of skill in this art, andinclude those that are used as a support matrix. These materialsinclude, but are not limited to, inorganics, natural polymers, andsynthetic polymers, including, but are not limited to: cellulose,cellulose derivatives, acrylic resins, glass, silica gels, polystyrene,gelatin, polyvinyl pyrrolidone, co-polymers of vinyl and acrylamide,polystyrene cross-linked with divinylbenzene or the like [see,Merrifield (1964) Biochemistry 3:1385-1390], polyacrylamides, latexgels, polystyrene, dextran, polyacrylamides, rubber, silicon, plastics,nitrocellulose, celluloses, natural sponges, and many others.

Among the preferred matrices are polymeric beads, such as the TENTAGEL™resins and derivatives thereof. [sold by Rapp Polymere, Tubingen,Germany; see, U.S. Pat. No. 4,908,405 and U.S. Pat. No. 5,292,814; see,also Butz et al. (1994) Peptide Res. 7:20-23: Kleine et al. (1994)Immunobiol. 190:53-66; see, also Piskin et al. (1994), Chapter 18“Nondegradable and Biodegradable Polymeric Particles” in DiagnosticBiosensor Polymers, ACS Symp. Series 556, Usmani et al. Eds, AmericanChemical Society, Washington, D.C.], which are designed for solid phasechemistry and for affinity separations and purifications. See, alsoBayer et al. (1994) in Pept.: Chem., Struct. Biol., Proc. Am. Pept.Symp., 13th; Hodges, et al. eds., pp. 156-158; Zhang et al. (1993) Pept.1992. Proc. Eur. Pept. Symp. 22nd, Schneider, et al., eds. pp. 432-433;Ilg et al. (1994) Macromolecules, pp. 2778-83; Zeppezauer et al. (1993)Z. Naturforsch., B: Chem. Sci. 48:1801-1806; Rapp et al. (1992) Pept.Chem. 1992. Proc. Jpn. Symp. 2nd, Yanaihara, ed., pp. 7-10; Nokihara etal. (1993) Shimadzu Hyoron 50:25-31; Wright et al. (1993) TetrahedronLett. 34:3373-3376; Bayer et al. (1992) Poly(Ethylene Glycol) Chem.Harris, ed., pp. 325-45; Rapp et al. (1990) Innovation Perspect. SolidPhase Synth. Collect. Pap., Int. Symp. 1st, Epton, ed., pp. 205-10; Rappet al. (1992) Pept.: Chem. Biol., Proc. Am. Pept. Symp. 12th, Smith etal., eds., pp. 529-530; Rapp et al. (1989) Pent., Proc. Eur. Pept. Symp.20th, Jung et al., ed., pp. 199-201; Bayer et al. (1986) Chem. Pept.Proteins 3: 3-8; Bayer et al. (1983) Pept.: Struct. Funct., Proc. Am.Pept. Symp. 8th, Hruby et al. eds., pp. 87-90 for descriptions ofpreparation of such beads and use thereof in synthetic chemistry.Matrices that are also contemplated for use herein includefluophore-containing or impregnated matrices, such as microplates andbeads [commercially available, for example, from Amersham, ArlingtonHeights, Ill.; plastic scintillation beads from NE (Nuclear Technology,Inc., San Carlos, Calif.), Packard, Meriden, Conn.]. It is understoodthat these commercially available materials will be modified bycombining them with memories, such as by methods described herein.

The matrix may also be a relatively inert polymer, which can be graftedby ionizing radiation [see, e.g., FIG. 21, which depicts a particularembodiment] to permit attachment of a coating of polystyrene or othersuch polymer that can be derivatized and used as a support. Radiationgrafting of monomers allows a diversity of surface characteristics to begenerated on plasmid supports [see, e.g., Maeji et al. (1994) ReactivePolymers 22:203-212; and Berg et al. (1989) J. Am. Chem. Soc.111:8024-8026]. For example, radiolytic grafting of monomers, such asvinyl monomers, or mixtures of monomers, to polymers, such aspolyethylene and polypropylene, produce composites that have a widevariety of surface characteristics. These methods have been used tograft polymers to insoluble supports for synthesis of peptides and othermolecules, and are of particular interest herein. The recording devices,which are often coated with a plastic or other insert material, can betreated with ionizing radiation so that selected monomers can be graftedto render the surface suitable for chemical syntheses.

Where the matrix particles are macroscopic in size, such as about atleast 1 mm in at least one dimension, such bead or matrix particle orcontinuous matrix, may contain one or more memories. Where the matrixparticles are smaller, such as NE particles [PVT-based plasticscintillator microsphere], which are about 1 to 10 μm in diameter, morethan one such particle will generally be associated with one memory.Also, the bead may include additional material, such as scintillant or afluophore impregnated therein. In preferred embodiments, the solid phasechemistry and subsequent assaying may be performed on the same bead ormatrix with memory combination. All procedures, including synthesis onthe bead and assaying and analysis, can be automated.

The matrices are typically insoluble substrates that are solid, porous,deformable, or hard, and have any required structure and geometry,including, but not limited to: beads, pellets, disks, capillaries,hollow fibers, needles, solid fibers, random shapes, thin films andmembranes. Typically, when the matrix is particulate, the particles areat least about 10-2000 μM, but may be smaller, particularly for use inembodiments in which more than one particle is in proximity to a memory.For purposes herein, the support material will typically encase or be incontact with the data storage device, and, thus, will desirably have atleast one dimension on the order of 1 mm [1000 μM] or more, althoughsmaller particles may be contacted with the data storage devices,particularly in embodiments in which more than one matrix particle isassociated, linked or in proximity to one memory or matrix with memory,such as the microvessels [see, e.g., FIGS. 11-16]. Each memory will bein associated with, in contact with or proximity to at least one matrixparticle, and may be in contact with more than one. As smallersemiconductor and electronic or optical devices become available, thecapacity of the memory can be increased and/or the size of the particlescan be decreased. For example, presently, 0.5 micron semiconductordevices are available. Integrated circuits 0.25-micron in size have beendescribed and are being developed using a technology called theComplementary Metal Oxide-Semiconductor process (see, e.g., Investor'sBusiness Daily May 30, 1995).

Also of interest herein, are devices that are prepared by inserting therecording device into a “tube” [see, e.g., FIG. 21] or encasing them inan inert material [with respect to the media in which the device will bein contact]. This material is fabricated from a plastic or other inertmaterial. Preferably prior to introducing [and preferably sealing] therecording device inside, the tube or encasing material is treated withionizing radiation to render the surface suitable for grafting selectedmonomers, such as styrene [see, e.g., Maeji et al. (1994) ReactivePolymers 22:203-212; and Berg et al. (1989) J. Am. Chem. Soc.111:8024-8026].

Recording device(s) is(are) introduced inside the material or thematerial is wrapped around the device and the resulting memory withmatrix “tubes” [MICROTUBES™, see, FIG. 21] are used for chemicalsynthesis or linkage of selected molecules or biological particles.These “tubes” are preferably synthesized from an inert resin, such as apolypropylene resin [a Moplen resin, V29G PP resin from Montell, NewarkDel., a distributor for Himont, Italy]. Any inert matrix that can thenbe functionalized or to which derivatizable monomers can be grafted issuitable. Preferably herein, polypropylene tubes are grafted and thenformed into tubes or other suitable shape and the recording deviceinserted inside. These tubes [MICROTUBES™] with grafted monomers arethen used as synthesis, and/or for assays or for multiplexed processes,including synthesis and assays or other multistep procedures.

Also larger matrix particles, which advantageously provide ease ofhandling, may be used and may be in contact with or proximity to morethan one memory [i.e., one particle may have a plurality of memories inproximity or linked to it; each memory may programmed with differentdata regarding the matrix particle, linked molecules, synthesis or assayprotocol, etc.]. Thus, so-called macro-beads (Rapp Polymere, Tubingen,Germany), which have a diameter of 2 mm when swollen, or other matricesof such size, are also contemplated for use herein. Particles of suchsize can be readily manipulated and the memory can be readilyimpregnated in or on the bead. These beads (available from Rapp) arealso advantageous because of their uniformity in size, which is usefulwhen automating the processes for electronically tagging and assayingthe beads.

The matrices may also include an inert strip, such as a teflon strip orother material to which the molecules or biological particles ofinterest do not adhere, to aid in handling the matrix, such asembodiments in which a matrix with memory and linked molecules orbiological particle are introduced into an agar-containing plate forimmunoassays or for antibiotic screening.

Selection of the matrices will be governed, at least in part, by theirphysical and chemical properties, such as solubility, functional groups,mechanical stability, surface area swelling propensity, hydrophobic orhydrophilic properties and intended use.

The data storage device with programmable memory may be coated with amaterial, such as a glass or a plastic, that can be further derivatizedand used as the support or it may be encased, partially or completely,in the matrix material, such as during or prior to polymerization of thematerial. Such coating may be performed manually or may be automated.The coating can be effected manually or using instruments designed forcoating such devices. Instruments for this purpose are available [see,e.g., the Series C3000 systems for dipping available from SpecialtyCoating Systems, Inc., Indianapolis, Ind.; and the Series CM 2000systems for spray coating available from Integrated Technologies, Inc.Acushnet, Mass.].

The data storage device with memory may be physically inserted into thematrix material or particle. It also can be manufactured with a coatingthat is suitable for use as a matrix or that includes regions in thecoating that are suitable for use as a matrix. If the matrix material isa porous membrane, it may be placed inside the membrane. It isunderstood that when the memory device is encased in the matrix orcoated with protective material, such matrix or material must betransparent to the signal used to program the memory for writing orreading data. More than one matrix particle may be linked to each datastorage device.

In some instances, the data storage device with memory is coated with apolymer, which is then treated to contain an appropriate reactive moietyor in some cases the device may be obtained commercially alreadycontaining the reactive moiety, and may thereby serve as the matrixsupport upon which molecules or biological particles are linked.Materials containing reactive surface moieties such as amino silanelinkages, hydroxyl linkages or carboxysilane linkages may be produced bywell established surface chemistry techniques involving silanizationreactions, or the like. Examples of these materials are those havingsurface silicon oxide moieties, covalently linked togamma-aminopropylsilane, and other organic moieties;N-[3-(triethyoxysilyl)propyl]phthelamic acid; and bis-(2-hydroxyethyl)aminopropyltriethoxysilane. Exemplary of readily available materialscontaining amino group reactive functionalities, include, but are notlimited to, para-aminophenyltriethyoxysilane. Also derivatizedpolystyrenes and other such polymers are well known and readilyavailable to those of skill in this art [e.g., the TENTAGEL® Resins areavailable with a multitude of functional groups, and are sold by RappPolymere, Tubingen, Germany; see, U.S. Pat. No. 4,908,405 and U.S. Pat.No. 5,292,814; see, also Butz et al. (1994) Peptide Res. 7:20-23; Kleineet al. (1994) Immunobiol. 190:53-66].

The data storage device with memory, however, generally should not orcannot be exposed to the reaction solution, and, thus, must be coatedwith at least a thin layer of a glass or ceramic or other protectivecoating that does not interfere with the operation of the device. Theseoperations include electrical conduction across the device andtransmission of remotely transmitted electromagnetic radiation by whichdata are written and read. It is such coating that may also serve as amatrix upon which the molecules or biological particles may be linked.

The data storage devices with memory may be coated either directly orfollowing coating with a ceramic, glass or other material, may then becoated with agarose, which is heated, the devices are dipped into theagarose, and then cooled to about room temperature. The resulting glass,silica, agarose or other coated memory device, may be used as the matrixsupports for chemical syntheses and reactions.

Conventional integrated circuit manufacturing and packaging methodsinclude methods and means for encapsulating integrated circuits toprotect the devices from the environment and to facilitate connection toexternal devices. Also, there are numerous descriptions for thepreparation of semiconductor devices and wires, particularly for use assensors [see, e.g., U.S. Pat. No. 4,933,285; see, also Cass, Ed. (1990)Biosensors A Practical Approach, IRL Press at Oxford University Press,Oxford; biosensors are chemosensors an can include a biologicaldetection system, generally biologically active substances, such asenzymes, antibodies, lectins and hormone receptors, which areimmobilized on the surface of the sensor electrode or in a thin layer onthe sensor electrode; biosensors are sensors that detect biologicalspecies], which measure electrochemical solution parameters, such as pH.Despite differences in the components of biosensors and recordingdevices used herein, certain of the methods for coating electrodes andwires in the biosensor art may be adapted for use herein [see, e.g.,U.S. Pat. Nos. 5,342,772, 5,389,534, 5,384,028, 5,296,122, 5,334,880,5,311,039, 4,777,019, 5,143,854, 5,200,051, 5,212,050, 5,310,686,5,324,591; see, also Usmani et al., ed. (1994) Diagnostic BiosensorPolymers, ACS Symposium Series No. 556].

It is, however, emphasized that the combinations herein of matrix withmemory are not sensors, which measure external parameters and caninclude electrodes that must be in contact with the solution such thatmolecules in solution directly contact the electrode, and which measuresolution parameters. Data regarding the combination, particularly thelinked or associated biological particle or matrix is written into thememory, and thus records information about itself. Sensors monitor whatis going outside of the device. The combinations herein of matrices withmemories can be enhanced by addition of sensor elements for themeasurement of external conditions, information about the externalconditions can be recorded into the combination's memory.

The combinations herein are matrix materials with recording devices thatcontain data storage units that include remotely programmable memories;the recording devices used in solution must be coated with a materialthat prevents contact between the recording device and the medium, suchas the solution or air or gas [e.g., nitrogen or oxygen or CO₂]. Theinformation is introduced into the memory by addressing the memory torecord information regarding molecules or biological particles linkedthereto. Except in the reaction detecting [verifying] embodiment, inwhich the memory can be encoded upon reaction of a linked molecule orbiological particle, solution parameters are not recorded in the memory.

In certain embodiments herein, the matrices with memories herein,however may be combined with devices or components or biosensors orother such sensor devices and used in connection therewith to monitorsolution or external parameters. For example, the combination may beelectronically or otherwise linked to a biosensor and informationobtained by the biosensor can be encoded in memory, or the combinationcan transmit information to the biosensor or, when used internally in ananimal, to monitor the location of a biosensor or to transmitinformation from the biosensor. For example, transponder memory devicesexemplified herein, include circuitry for measuring and recordingsolution temperature. These transponders can be modified to read andrecord pH, instead of or in addition to temperature. Thus, duringsynthesis or other processing steps of linked or proximate molecules orbiological particles, RF or other EM radiation will be used to encodeinformation in the memory and at the same time pH and/or temperature inthe external solution can be measured and recorded in the memory.

1. Natural Matrix Support Materials

Naturally-occurring supports include, but are not limited to agarose,other polysaccharides, collagen, celluloses and derivatives thereof,glass, silica, and alumina. Methods for isolation, modification andtreatment to render them suitable for use as supports is well known tothose of skill in this art [see, e.g., Hermanson et al. (1992)Immobilized Affinity Ligand Techniques, Academic Press, Inc., SanDiego]. Gels, such as agarose, can be readily adapted for use herein.Natural polymers such as polypeptides, proteins and carbohydrates;metalloids, such as silicon and germanium, that have semiconductiveproperties, as long as they do not interfere with operation of the datastorage device may also be adapted for use herein. Also, metals such asplatinum, gold, nickel, copper, zinc, tin, palladium, silver, again aslong as the combination of the data storage device with memory, matrixsupport with molecule or biological particle does not interfere withoperation of the device with memory, may be adapted for use herein.Other matrices of interest include oxides of the metal and metalloidssuch as Pt—PtO, Si—SiO, Au—AuO, TiO2, Cu—CuO, and the like. Alsocompound semiconductors, such as lithium niobate, gallium arsenide andindium-phosphide, and nickel-coated mica surfaces, as used inpreparation of molecules for observation in an atomic force microscope[see, e.g., Ill et al. (1993) Biophys J. 64:919] may be used asmatrices. Methods for preparation of such matrix materials are wellknown.

For example, U.S. Pat. No. 4,175,183 describes a water insolublehydroxyalkylated cross-linked regenerated cellulose and a method for itspreparation. A method of preparing the product using near stoichiometricproportions of reagents is described. Use of the product directly in gelchromatography and as an intermediate in the preparation of ionexchangers is also described.

2. Synthetic Matrices

There are innumerable synthetic matrices and methods for theirpreparation known to those of skill in this art. Synthetic matrices aretypically produced by polymerization of functional matrices, orcopolymerization from two or more monomers of from a synthetic monomerand naturally occurring matrix monomer or polymer, such as agarose.Before such polymers solidify, they are contacted with the data storagedevice with memory, which can be cast into the material or dipped intothe material. Alternatively, after preparation of particles or largersynthetic matrices, the recording device containing the data storageunit(s) can be manually inserted into the matrix material. Again, suchdevices can be pre-coated with glass, ceramic, silica or other suitablematerial.

Synthetic matrices include, but are not limited to: acrylamides,dextran-derivatives and dextran co-polymers, agarose-polyacrylamideblends, other polymers and co-polymers with various functional groups,methacrylate derivatives and co-polymers, polystyrene and polystyrenecopolymers [see, e.g., Merrifield (1964) Biochemistry 3:1385-1390; Berget al. (1990) in Innovation Perspect. Solid Phase Synth. Collect. Pap.,Int. Symp., 1st, Epton, Roger (Ed), pp. 453-459; Berg et al. (1989) inPept., Proc. Eur. Pept. Symp., 20th, Jung, G. et al. (Eds), pp. 196-198;Berg et al. (1989) J. Am. Chem. Soc. 111:8024-8026; Kent et al. (1979)Isr. J. Chem. 17:243-247; Kent et al. (1978) J. Org. Chem. 43:2845-2852;Mitchell et al. (1976) Tetrahedron Lett. 42:3795-3798; U.S. Pat. No.4,507,230; U.S. Pat. No. 4,006,117; and U.S. Pat. No. 5,389,449].Methods for preparation of such matrices are well-known to those ofskill in this art.

Synthetic matrices include those made from polymers and co-polymers suchas polyvinylalcohols, acrylates and acrylic acids such aspoly-ethylene-co-acrylic acid, polyethylene-co-methacrylic acid,polyethylene-co-ethylacrylate, polyethylene-co-methyl acrylate,polypropylene-co-acrylic acid, polypropylene-co-methyl-acrylic acid,polypropylene-co-ethylacrylate, polypropylene-co-methyl acrylate,polyethylene-co-vinyl acetate, polypropylene-co-vinyl acetate, and thosecontaining acid anhydride groups such as polyethylene-co-maleicanhydride, polypropylene-co-maleic anhydride and the like. Liposomeshave also been used as solid supports for affinity purifications [Powellet al. (1989) Biotechnol. Bioeng. 33:173].

For example, U.S. Pat. No. 5,403,750, describes the preparation ofpolyurethane-based polymers. U.S. Pat. No. 4,241,537 describes a plantgrowth medium containing a hydrophilic polyurethane gel compositionprepared from chain-extended polyols; random copolymerization ispreferred with up to 50% propylene oxide units so that the prepolymerwill be a liquid at room temperature. U.S. Pat. No. 3,939,123 describeslightly crosslinked polyurethane polymers of isocyanate terminatedprepolymers containing poly(ethyleneoxy) glycols with up to 35% of apoly(propyleneoxy) glycol or a poly(butyleneoxy) glycol. In producingthese polymers, an organic polyamine is used as a crosslinking agent.Other matrices and preparation thereof are described in U.S. Pat. Nos.4,177,038, 4,175,183, 4,439,585, 4,485,227, 4,569,981, 5,092,992,5,334,640, 5,328,603

U.S. Pat. No. 4,162,355 describes a polymer suitable for use in affinitychromatography, which is a polymer of an aminimide and a vinyl compoundhaving at least one pendant halo-methyl group. An amine ligand, whichaffords sites for binding in affinity chromatography is coupled to thepolymer by reaction with a portion of the pendant halo-methyl groups andthe remainder of the pendant halo-methyl groups are reacted with anamine containing a pendant hydrophilic group. A method of coating asubstrate with this polymer is also described. An exemplary aminimide is1,1-dimethyl-1-(2-hydroxyoctyl)amine methacrylimide and vinyl compoundis a chloromethyl styrene.

U.S. Pat. No. 4,171,412 describes specific matrices based on hydrophilicpolymeric gels, preferably of a macroporous character, which carrycovalently bonded D-amino acids or peptides that contain D-amino acidunits. The basic support is prepared by copolymerization of hydroxyalkylesters or hydroxyalkylamides of acrylic and methacrylic acid withcrosslinking acrylate or methacrylate comonomers are modified by thereaction with diamines, aminoacids or dicarboxylic acids and theresulting carboxyterminal or aminoterminal groups are condensed withD-analogs of aminoacids or peptides. The peptide containing D-aminoacidsalso can be synthesized stepwise on the surface of the carrier.

U.S. Pat. No. 4,178,439 describes a cationic ion exchanger and a methodfor preparation thereof. U.S. Pat. No. 4,180,524 describes chemicalsyntheses on a silica support.

Immobilized Artificial Membranes [IAMs; see, e.g., U.S. Pat. Nos.4,931,498 and 4,927,879] may also be used. IAMs mimic cell membraneenvironments and may be used to bind molecules that preferentiallyassociate with cell membranes [see, e.g., Pidgeon et al. (1990) EnzymeMicrob. Technol. 12:149].

3. Immobilization and Activation

Numerous methods have been developed for the immobilization of proteinsand other biomolecules onto solid or liquid supports [see, e.g., Mosbach(1976) Methods in Enzymology 44; Weetall (1975) Immobilized Enzymes,Antigens, Antibodies, and Peptides; and Kennedy et al. (1983) SolidPhase Biochemistry, Analytical and Synthetic Aspects, Scouten, ed., pp.253-391; see, generally, Affinity Techniques. Enzyme Purification: PartB. Methods in Enzymology, Vol. 34, ed. W. B. Jakoby, M. Wilchek, Acad.Press, N.Y. (1974); Immobilized Biochemicals and AffinityChromatography, Advances in Experimental Medicine and Biology, vol. 42,ed. R. Dunlap, Plenum Press, N.Y. (1974)].

Among the most commonly used methods are absorption and adsorption orcovalent binding to the support, either directly or via a linker, suchas the numerous disulfide linkages, thioether bonds, hindered disulfidebonds, and covalent bonds between free reactive groups, such as amineand thiol groups, known to those of skill in art [see, e.g., the PIERCECATALOG, ImmunoTechnology Catalog & Handbook, 1992-1993, which describesthe preparation of and use of such reagents and provides a commercialsource for such reagents; and Wong (1993) Chemistry of ProteinConjugation and Cross Linking, CRC Press; see, also DeWitt et al. (1993)Proc. Natl. Acad. Sci. U.S.A. 90:6909; Zuckermann et al. (1992) J. Am.Chem. Soc. 114:10646; Kurth et al. (1994) J. Am. Chem. Soc. 116:2661;Ellman et al. (1994) Proc. Natl. Acad. Sci. U.S.A. 91:4708; Sucholeiki(1994) Tetrahedron Lttrs. 35:7307; and Su-Sun Wang (1976) J. Org. Chem.41:3258; Padwa et al. (1971) J. Org. Chem. 41:3550 and Vedejs et al.(1984) J. Org. Chem. 49:575, which describe photosensitive linkers]

To effect immobilization, a solution of the protein or other biomoleculeis contacted with a support material such as alumina, carbon, anion-exchange resin, cellulose, glass or a ceramic. Fluorocarbon polymershave been used as supports to which biomolecules have been attached byadsorption [see, U.S. Pat. No. 3,843,443; Published International PCTApplication WO/86 03840].

A large variety of methods are known for attaching biological molecules,including proteins and nucleic acids, molecules to solid supports [seee.g., U.S. Pat. No. 5,451,683]. For example, U.S. Pat. No. 4,681,870describes a method for introducing free amino or carboxyl groups onto asilica matrix. These groups may subsequently be covalently linked toother groups, such as a protein or other anti-ligand, in the presence ofa carbodiimide. Alternatively, a silica matrix may be activated bytreatment with a cyanogen halide under alkaline conditions. Theanti-ligand is covalently attached to the surface upon addition to theactivated surface. Another method involves modification of a polymersurface through the successive application of multiple layers of biotin,avidin and extenders [see, e.g., U.S. Pat. No. 4,282,287]; other methodsinvolve photoactivation in which a polypeptide chain is attached to asolid substrate by incorporating a light-sensitive unnatural amino acidgroup into the polypeptide chain and exposing the product to low-energyultraviolet light [see, e.g., U.S. Pat. No. 4,762,881]. Oligonucleotideshave also been attached using a photochemically active reagents, such asa psoralen compound, and a coupling agent, which attaches thephotoreagent to the substrate [see, e.g., U.S. Pat. No. 4,542,102 andU.S. Pat. No. 4,562,157]. Photoactivation of the photoreagent binds anucleic acid molecule to the substrate to give a surface-bound probe.

Covalent binding of the protein or other biomolecule or organic moleculeor biological particle to chemically activated solid matrix supportssuch as glass, synthetic polymers, and cross-linked polysaccharides is amore frequently used immobilization technique. The molecule orbiological particle may be directly linked to the matrix support orlinked via linker, such as a metal [see, e.g., U.S. Pat. No. 4,179,402;and Smith et al. (1992) Methods: A Companion to Methods in Enz.4:73-78]. An example of this method is the cyanogen bromide activationof polysaccharide supports, such as agarose. The use of perfluorocarbonpolymer-based supports for enzyme immobilization and affinitychromatography is described in U.S. Pat. No. 4,885,250]. In this methodthe biomolecule is first modified by reaction with a perfluoroalkylatingagent such as perfluorooctylpropylisocyanate described in U.S. Pat. No.4,954,444. Then, the modified protein is adsorbed onto the fluorocarbonsupport to effect immobilization.

The activation and use of matrices are well known and may be effected byany such known methods [see, e.g., Hermanson et al. (1992) ImmobilizedAffinity Ligand Techniques, Academic Press, Inc., San Diego]. Forexample, the coupling of the amino acids may be accomplished bytechniques familiar to those in the art and provided, for example, inStewart and Young, 1984, Solid Phase Synthesis, Second Edition, PierceChemical Co., Rockford.

Molecules may also be attached to matrices through kinetically inertmetal ion linkages, such as Co(III), using, for example, native metalbinding sites on the molecules, such as IgG binding sequences, orgenetically modified proteins that bind metal ions [see, e.g., Smith etal. (1992) Methods: A Companion to Methods in Enzymology 4, 73 (1992);Ill et al. (1993) Biophys J. 64:919′ Loetscher et al. (1992) J.Chromatography 595:113-199; U.S. Pat. No. 5,443,816; Hale (1995)Analytical Biochem. 231:46-49].

Other suitable methods for linking molecules and biological particles tosolid supports are well known to those of skill in this art [see, e.g.,U.S. Pat. No. 5,416,193]. These linkers include linkers that aresuitable for chemically linking molecules, such as proteins and nucleicacid, to supports include, but are not limited to, disulfide bonds,thioether bonds, hindered disulfide bonds, and covalent bonds betweenfree reactive groups, such as amine and thiol groups. These bonds can beproduced using heterobifunctional reagents to produce reactive thiolgroups on one or both of the moieties and then reacting the thiol groupson one moiety with reactive thiol groups or amine groups to whichreactive maleimido groups or thiol groups can be attached on the other.Other linkers include, acid cleavable linkers, such asbismaleimideothoxy propane, acid labile-transferrin conjugates andadipic acid diihydrazide, that would be cleaved in more acidicintracellular compartments; cross linkers that are cleaved upon exposureto UV or visible light and linkers, such as the various domains, such asC_(H)1, C_(H)2, and C_(H)3, from the constant region of human IgG₁ (see,Batra et al. (1993) Molecular Immunol. 30:379-386).

Presently preferred linkages are direct linkages effected by adsorbingthe molecule or biological particle to the surface of the matrix. Otherpreferred linkages are photocleavable linkages that can be activated byexposure to light [see, e.g., Baldwin et al. (1995) J. Am. Chem. Soc.117:5588; Goldmacher et al. (1992) Bioconj. Chem. 3:104-107, whichlinkers are herein incorporated by reference]. The photocleavable linkeris selected such that the cleaving wavelength that does not damagelinked moieties. Photocleavable linkers are linkers that are cleavedupon exposure to light [see, e.g., Hazum et al. (1981) in Pept., Proc.Eur. Pept. Symp. 16th, Brunfeldt, K (Ed), pp. 105-110, which describesthe use of a nitrobenzyl group as a photocleavable protective group forcysteine; Yen et al. (1989) Makromol. Chem. 190:69-82, which describeswater soluble photocleavable copolymers, includinghydroxypropylmethacrylamide copolymer, glycine copolymer, fluoresceincopolymer and methylrhodamine copolymer; Goldmacher et al. (1992)Bioconj. Chem. 3:104-107, which describes a cross-linker and reagentthat undergoes photolytic degradation upon exposure to near UV light(350 nm); and Senter et al. (1985) Photochem. Photobiol 42:231-237,which describes nitrobenzyloxycarbonyl chloride cross linking reagentsthat produce photocleavable linkages]. Other linkers include fluoridelabile linkers [see, e.g., Rodolph et al. (1995) J. Am. Chem. Soc.117:5712], and acid labile linkers [see, e.g., Kick et al. (1995) J.Med. Chem. 38:1427]. The selected linker will depend upon the particularapplication and, if needed, may be empirically selected.

B. Data Storage Units with Memory

Any remotely programmable data storage device that can be linked to orused in proximity to the solid supports and molecules and biologicalparticles as described herein is intended for use herein. Preferreddevices are rapidly and readily programmable using penetratingelectromagnetic radiation, such as radio frequency or visible lightlasers, operate with relatively low power, have fast access [preferably1 sec or less, more preferably 10²-10³ sec], and are remotelyprogrammable so that information can be stored or programmed and laterretrieved from a distance, as permitted by the form of theelectromagnetic signal used for transmission. Presently preferreddevices are on the order of 1-10 mm in the largest dimension and areremotely programmable using RF or radar.

Recording devices may be active, which contain a power source, such as abattery, and passive, which does not include a power source. In apassive device, which has no independent power source, thetransmitter/receiver system, which transfers the data between therecording device and a host computer and which is preferably integratedon the same substrate as the memory, also supplies the power to programand retrieve the data stored in the memory. This is effected byintegrating a rectifier circuit onto the substrate to convert thereceived signal into an operating voltage.

Alternatively, an active device can include a battery [see, e.g., U.S.Pat. No. 5,442,940, U.S. Pat. No. 5,350,645, U.S. Pat. No. 5,212,315,U.S. Pat. No. 5,029,214, U.S. Pat. No. 4,960,983] to supply the power toprovide an operating voltage to the memory device. When a battery isused the memory can be an EEPROM, a DRAM, or other erasable memoryrequiring continuous power to retain information. It may be desirable tocombine the antenna/rectifier circuit combination with a battery tocreate a passive/active device, with the voltages supplied by eachsource supplementing each other. For example, the transmitted signalcould provide the voltage for writing and reading, while the battery, inaddition to supplementing this voltage, provides a refresh voltage for aDRAM memory so that data is retained when the transmitted signal isremoved.

The remotely programmable device can be programmed sequentially to beuniquely identifiable during and after stepwise synthesis ofmacromolecules or before, or during, or after selection of screenedmolecules. In certain embodiments herein, the data storage units areinformation carriers in which the functions of writing data and readingthe recorded data are empowered by an electromagnetic signal generatedand modulated by a remote host controller. Thus, the data storagedevices are inactive, except when exposed to the appropriateelectromagnetic signal. In an alternative embodiment, the devices may beoptically or magnetically programmable read/write devices.

Electromagnetically Programmable Devices

The programmable devices intended for use herein, include any devicethat can record or store data. The preferred device will be remotelyprogrammable and will be small, typically on the order of 10-20 mm³ [or10-20 mm in its largest dimension] or, preferably smaller. Any means forremote programming and data storage, including semiconductors andoptical storage media are intended for use herein.

Also intended for use herein, are commercially available precodeddevices, such as identification and tracking devices for animals andmerchandise, such those used with and as security systems [see, e.g.,U.S. Pat. Nos. 4,652,528, 5,044,623, 5,099,226, 5,218,343, 5,323,704,4,333,072, 4,321,069, 4,318,658, 5,121,748, 5,214,409, 5,235,326,5,257,011 and 5,266,926], and devices used to tag animals. These devicesmay also be programmable using an RF signal. These device can bemodified, such as by folding it, to change geometry to render them moresuitable for use in the methods herein. Of particular interest hereinare devices sold by BioMedic Data Systems, Inc, NJ [see, e.g., theIPTT-100 purchased from BioMedic Data Systems, Inc., Maywood, N.J.; see,also U.S. Pat. Nos. 5,422,636, 5,420,579, 5,262,772, 5,252,962,5,250,962, and see, also, U.S. application Ser. No. 08/322,644, filedOct. 13, 1994]. ID tags available from IDTAG™ Inc, particularly theIDT150 read/write transponder [ITDAG™ Ltd. Bracknell, Berks RG12 3XQ,UK, fabricated using standard procedures and the method for coilwinding, bonding and packaging described in International PCTapplication Nos. WO 95/33246, WO 95/16270, WO 94/24642, WO 93/12513. WO92/15105, WO 91/16718; see, also U.S. Pat. Nos. 5,223,851 and 5,281,855]are also preferred herein. The IDT150 is a CMOS device that provides akilobit of EEROM. This transponder also includes a 32 bit fixed codeserial number that uniquely identifies each chip. The IDTAG™ transpondertransmits data to a transceiver system by amplitude modulating its coiland generating an EM field. It receives data and commands from atransceiver by demodulating the field received by the coil and decodingthe commands. The transponder derives its power source from a frequencyemitted in the signal from the reader, to which the transponder emits aresponse. A smaller version [that has 16 bit EEROM] and is about 11 mm×4mm×3 mm of this transponder is also among preferred devices. Thesetransponders are packaged in glass or polystyrene or other suchmaterial.

In a preferred embodiment herein, the data storage unit includes asemiconductor chip with integrated circuits formed thereon including amemory and its supporting circuitry. These devices can be written to andinterrogated from a distance. A radio frequency transmitter/receiversystem supplies power to program and retrieve data. In particular, thedata storage unit preferably includes a programmable read onlysemiconductor memory [PROM], preferably a non-volatile memory or othermemory that can store data for future retrieval, that will haveinformation describing or identifying the molecules or biologicalparticles linked to or in proximity to the matrix. This informationeither identifies the molecule or biological particles including a phageand viral particles, bacteria, cells and fragments thereof, provides ahistory of the synthesis of the molecule, or provides information, suchas a batch number, quality control data, reaction number, and/oridentity of the linked entity. The memory is programmed, before, duringor, preferably, after, each step of synthesis and can thereafter beread, thereby identifying the molecule or its components and order ofaddition, or process of synthesis.

While many well known read only memory devices use fuse structures thatare selectively “blown” to store data points, with a fuse located ateach possible data address in an array, among the devices of interestherein are those that rely on antifuse programming technology, in whichshort circuits are selectively created through an insulating layerseparating word and bit lines in an array. Due to the relatively lowlevel of voltage supplied by the transmitted signal when the memorydevice is passive, antifuse memories are readily used because of thelower voltage requirements for writing.

Thus, suitable memory devices, are about 1-20 mm in the smallestdimension [or smaller], are rapidly programmable [1 sec, preferably 1msec or less], can be interrogated from a distance [distances of about acentimenter up to about an inch are presently preferred], and areprogrammable using electromagnetic radiation, preferably frequencies,such as those within the radio frequency range, that do not alter theassessed activities and physical properties of the molecules andbiological particles of interest.

Devices that rely on other programmable volatile memories are alsointended for use herein. For example, a battery may be used as to supplythe power to provide an operating voltage to the memory device. When abattery is used the memory can be an EEPROM, a DRAM, or other erasablememory requiring continuous power to retain information. It may beadvantageous to combine the antenna/rectifier circuitry with a batteryto create a passive/active device, in which the voltages supplied byeach source supplement each other. For example, the transmitted signalcould provide the voltage for writing and reading, while the battery, inaddition to supplementing this write/read voltage, provides a refreshvoltage for a DRAM memory so that data is retained when the transmittedsignal is removed.

Antifuses

An antifuse contains a layer of antifuse material sandwiched between twoconductive electrodes. The antifuse device is initially an opencircuited device in its unprogrammed state and can be irreversiblyconverted into an essentially short circuited device by the applicationof a programming voltage across the two electrodes to disrupt theantifuse material and create a low resistance current path between thetwo electrodes.

An exemplary antifuse structure for use herein is formed by defining aword line of heavily N-doped polysilicon on an insulating substrate,depositing an antifuse layer of lightly N-doped semiconductor over thepolysilicon, and defining a metal address [or bit] line upon and inelectrical contact with the antifuse layer. The semiconductor materialused for the antifuse layer is typically selected from among silicon,germanium, carbon and alpha-tin. The properties of the semiconductormaterial are such that the material is essentially non-conductive aslong as the voltage across it does not exceed a threshold level. Oncethe threshold voltage is exceeded, a conductive filament is formedthrough the semiconductor so that the resistance between the metal andpolysilicon lines at the points at which they cross irreversiblyswitches from a high resistance state to a relatively low resistancestate.

To program or change the resistance of the antifuse from a very highlevel [greater than 100,000,000 ohms] to a low level [less than 1000ohms], a voltage of sufficiently high electrical field strength isplaced across the antifuse film to create a short circuit. The voltagelevel required to induce breakdown is determined by the level of dopantin the antifuse layer. As breakdown occurs electrical current will flowthrough one small region of the film. The current is limited by theresistance of the filament itself as well as any series resistance ofconductive layers or logic devices [transistors] in series with theantifuse.

Examples of the antifuse and its use as a memory cell within a Read-OnlyMemory are discussed in Roesner et al., “Apparatus and Method of Use ofRadio frequency Identification Tags”, U.S. application Ser. No.08/379,923, filed Jan. 27, 1995, Roesner, “Method of Fabricating a HighDensity Programmable Read-Only Memory”, U.S. Pat. No. 4,796,074 (1989)and Roesner, “Electrically Programmable Read-Only Memory Stacked above aSemiconductor Substrate”, U.S. Pat. No. 4,442,507 (1984). A preferredantifuse is described in U.S. Pat. No. 5,095,362. “Method for reducingresistance for programmed antifuse” (1992) [see, also U.S. Pat. Nos.5,412,593 and 5,384,481].

U.S. Pat. No. 5,095,362 provides a method for fabricating a layer ofprogrammable material within an antifuse that exhibits relatively lowerthan normal resistance in its programmed state and also provides asemiconductor device containing an antifuse film of the type composed ofsemiconductor material having a first electrical state that ischaracterized by high electrical resistivity and a second electricalstate that is characterized by low electrical resistivity.

The means for selectively decreasing resistivity includes nonactivatedconductive dopants that are ion implanted within the otherwise highlyresistive semiconductor material. The dopants as implanted are in anonactivated state so that the dopants do not enhance the conduction ofcarriers in the film. Once activated, the dopants enhance the conductionof carriers in the film. Activation of the dopants occurs uponapplication of a threshold voltage across a predetermined and selectedportion of the material in which the dopants are disposed. The selectedportion is defined by the crossover point of selected word and bit [oraddress] lines. The dopants are N-type, selected from among antimony,phosphorous, arsenic, and others to provide additional charge carriers.The implant dosage is used to determine the threshold voltage level thatwill be required to induce formation of the conductive filament. P-typedopants, such as boron, may also be used to affect a change inprogramming voltage.

A Preferred Recording Device with Non-Volatile, Such as Anti-Fuse-Based,Memory

Referring to FIG. 5, which depicts a preferred embodiment, a recordingdevice containing a non-volatile electrically-programmable read-onlymemory [ROM] 102 that utilizes antifuse technology [or EEPROM or othersuitable memory] is combined on a single substrate 100 with a thin-filmplanar antenna 110 for receiving/transmitting an RF signal 104, arectifier 112 for deriving a voltage from a received radio frequency[RF] signal, an analog-to-digital converter [ADC] 114 for converting thevoltage into a digital signal for storage of data in the memory, and adigital-to-analog converter [DAC] 116 for converting the digital datainto a voltage signal for transmission back to the host computer isprovided. A single substrate 100 is preferred to provide the smallestpossible chip, and to facilitate encapsulation of the chip with aprotective, polymer shell [or shell+matrix or matrix material] 90. Shell90 must be non-reactive with and impervious to the various processesthat the recording device is being used to track in order to assure theintegrity of the memory device components on the chip. Materials for theshell include any such materials that are known to those of skill in theart [see, e.g., Hiroshi et al., eds. (1995) Polymeric Materials forMicroelectronic Applications: Science and Technology, ACS SymposiumSeries No. 579], including glasses, ceramics, plastics and other inertcoatings.

Based on current semiconductor integrated circuit fabrication processcapabilities, in a preferred embodiment the finished chip on which allof the listed components are integrated is on the order of 1 mm×1 mm[˜40 mils×40 mils], with a memory capacity of about 1024 bits, but canhave greater or lesser capacity as required or desired. Greater memorycapacity, where needed, and smaller chips, however, will be preferred.The chip may be larger to accommodate more memory if desired, or may besmaller as design rules permit smaller transistors and higher devicedensities, i.e., greater memory capacity.

The antifuse ROM structure described herein, and the method forfabricating the same, are based upon the teachings of U.S. Pat. No.4,424,579, issued Jan. 3, 1984, No. 4,442,507, issued Apr. 10, 1984, No.4,796,074, issued Jan. 3, 1989, and No. 5,095,362, issued Mar. 10, 1992,all of Roesner, No. 4,598,386, issued Jul. 1, 1986, of Roesner et al.,and No. 5,148,256, issued Sep. 15, 1992 and No. 5,296,722, issued Mar.22, 1994, both of Potash, et al., and also U.S. application Ser. No.08/379,923, filed Jan. 27, 1995, to Roesner et al., all of which areincorporated herein by reference.

In an antifuse-type memory device, the individual memory cells arearranged in arrays of orthogonal conductive word and bit lines to obtainthe smallest possible memory array size. For example, for 1024 bits ofmemory, there are 32 word lines and 32 bit lines for a square array.Memories with greater capacity may also be used. Schottky diodes areformed generally corresponding to the points at which the word and bitlines cross. The word and bit lines are separated by an undoped orlightly-doped semiconductor layer with interstitial doping. Thesemiconductor layer may also be amorphous silicon with implanted dopantsin a nonactivated state. Each of these crossover points is a memory celland is the equivalent of a programmable switch in series with a Schottkydiode. Data are stored by the switch being ON or OFF. As fabricated, anantifuse memory device has all of its switches in the OFF state. Aswitch is turned on by applying a voltage in excess of a pre-determinedthreshold voltage to one of the word lines while setting a selected bitline to a low logic level. The threshold voltage is determined by theimpedance of the semiconductor layer, i.e., its doping level. Accordingto the process for fabricating the antifuse memory of the preferredembodiment, the impedance can be less than 200 ohms with a thresholdvoltage for programming as low as 3 volts. Since in the embodimentdescribed herein the programming voltage is provided solely by therectified RF signal, a low threshold is preferred. Application ofvoltage exceeding the threshold activates the interstitial dopant in thesemiconducting film at the point corresponding to the cross-over betweenthe two lines, causing a short between the word and bit lines andirreversibly turning on that particular switch or memory cell. Addressdecoders, as are known in the art, are used to selectively address theword and bit lines for purposes of both writing information to andreading stored information from the memory array. [See, e.g., U.S. Pat.Nos. 5,033,623, 5,099,226, 5,105,190, 5,218,343, 5,323,704]. Exemplarymeans for decoding information to be stored in memory and to be readfrom memory are provided in U.S. Pat. No. 4,442,507 and U.S. Pat. No.4,598,386.

Information to be written into the memory need not be detailed since thedata stored in the memory is primarily acting as an identificationmarker that is traceable to a more detailed record stored in the hostcomputer memory 120, independent of the memory associated with thematrix support or tagged molecule or biological particle. In thismanner, the RF signal from transmitter 80 that is used to provide thepower and the signal to the matrix particle memory need only address asingle memory cell to indicate that a nascent oligomer linked to or inproximity to the memory device has been subjected to a given processstep or to identify a molecule or biological particle. In other words, aconventional “push-pull” type of address decoder, where only one bitline and one word line are driven high and low, respectively, at anygiven time, may be used. Thus, a sophisticated memory addressing systemneed not be provided on the matrix particle memory chip, and shiftregisters may be used to control memory addressing. Alternatively, amicroprocessor which is mask-programmed during the fabrication processfor controlling an address bus which connects the ADC 114 and the DAC116 to the memory array may also be built onto the same substrate onwhich the memory and other components are integrated. Other integratedmeans for selectively addressing locations within the memory are knownand will be apparent to the practitioner skilled in the art.

As described above, antifuse memories are well known in the art. Thesememories include structures in which the word and bit lines may both bemade of either N+ polysilicon or metal [aluminum or aluminum-silicon],separated by silicon dioxide (SiO₂), silicon nitride (Si₃N₄),combinations thereof, or amorphous silicon alone or in combination withSiO₂ and/or Si₃N₄. In each case, a short circuit is created at locationsin the antifuse material corresponding to the crossover location ofselected word and bit lines by applying a voltage in excess of apre-determined threshold voltage.

Examples of alternate means for forming an antifuse memory are providedin the following U.S. Patents: U.S. Pat. No. 5,248,632, issued Sep. 28,1993, of Tung et al.; U.S. Pat. No. 5,250,459, issued Oct. 5, 1993, ofLee, U.S. Pat. No. 5,282,158, issued Jan. 25, 1994, of Lee; U.S. Pat.No. 5,290,734, issued Mar. 1, 1994, of Boardman, et al.; U.S. Pat. No.5,300,456, issued Apr. 5, 1994, of Tigelaar et al.; U.S. Pat. No.5,311,039, issued May 10, 1994, of Kimura, et al.; U.S. Pat. No.5,316,971, issued May 31, 1994, of Chiang et al.; U.S. Pat. No.5,322,812, issued Jun. 21, 1994, of Dixit, et al.; No. 5,334,880, issuedAug. 2, 1994, of Abadeer, et al., and others.

Generally for use in the methods herein, non-volatility of the memory orthe ability to lock or prevent erasure is preferred since power isapplied to the chip only when it is subjected to the RF or othertransmission signal for reading or reading and writing. Furtherconsiderations are the voltage levels required for writing into memory,since the threshold voltage must be less than the maximum voltage of therectified RF signal in order to assure that sufficient voltage is alwaysavailable during the writing process. The write voltage may be enhancedby supplementing the RF-supplied voltage with optically-generatedvoltage, such as a photocell. Photocells on semiconductor substrates arewell known in the art and could be easily integrated onto the chip. Alaser or other light source could be readily included in the writeapparatus to illuminate the chip coincident with transmission of the RFwrite signal. Similarly, other forms of electromagnetic radiation may beused to provide additional power, if needed.

Although antifuse memories are not designed to be erasable, it may bedesirable to re-use the devices if the memory becomes full. In suchinstances, conventional electrically programmable erasable read onlymemories [EEPROMs] may be used instead. Since EEPROMs require higherwrite voltage levels, it may be desirable to supplement the RF-suppliedvoltage as described above. In EEPROMs, stored data can be erased byexposing the device to UV light.

Signal rectifier 112 may be one or more Schottky diode(s), making itreadily incorporated into the fabrication process used for the memoryarray. Other means for signal rectification may be used as are known.The ADC 114 and DAC 116 are well-known devices and are readilyintegrated onto the substrate 100 using the fabrication processdescribed in the references for the memory array. Radio frequencymodulation techniques, which are known in the art, for example, pulsecode modulation, may be adapted to permit direct digital transmission,in which case the ADC and DAC may not be required.

Antenna 110 is formed during the fabrication process using conventionalphotolithographic techniques to provide one or more metal structures,such as aluminum, to receive a pre-determined wavelength RFtransmission. The antenna may be a simple straight line half-waveantenna which is created by patterning a structure during the secondmetal process steps so that the structure has a length equal to one-halfof the wavelength of the selected RF transmission frequency in freespace. Another option for formation of the antenna is as a small loop,either on a dedicated portion of the chip, or encircling the othercomponents of the chip, also formed during the second metal step of thefabrication process. It is noted that, in a typical semiconductorfabrication process, such as would be compatible with the preferredantifuse memory, the first and second metal steps include depositing alayer of aluminum, then patterning the aluminum photolithographicallyfollowed by a plasma etch to define the desired features. Except wherevias are formed, the two metal layers are separated by a dielectricfilm. Dipole antennas may be formed by patterning the second metal in asimilar manner, with the dimensions of the antenna being selected forthe appropriate RF frequency. The two metal layers may also be used toform a microstrip antenna structure by selecting the dielectric filmbetween the metal layers such that it has a dielectric constant andthickness appropriate so that the microstrip is resonant at one-half ofthe RF wavelength. [The first metal layer provides the ground plane.]The metal structures, which may be square patches, circles, lines, orother geometries, are defined photolithographically during the normalmasking steps of the first and second metal processes. Other antennastructures which can be configured as a thin film device for integrationonto a common substrate with the memory structure and other componentsmay be used and will be apparent to those skilled in the art. Similarly,a resonant circuit [inductor-capacitor] can be readily integrated ontothe chip, with the resonant circuit being tuned to the RF carrier signalof the transmitter.

Frequency tuning of either an antenna or resonant circuit can provideadditional coding capability. For example, a first group of memorydevices can be tuned to receive a carrier wave of a first RF frequency,e.g., f₁, and a second group could be tuned to receive a secondfrequency f₂, and so on. The separate carrier frequencies could provideadditional means for tracking or providing information to the devices,even if the groups become intermixed.

The RF antenna may, in an alternate embodiment, be formed external tothe semiconductor substrate. In this configuration, a separateconductive wire, which acts as an antenna, will be attached to a bondpad formed on the chip using methods known to those skilled in the art.The wire will then be stabilized when the chip is encased in theprotective shell, so that the antenna extends at some angle to the chip.

Also, as an alternative to signal transmission via RF, the antifuse orother semiconductor memory and supporting circuitry can receive theaddressing commands and device power by optical transmission. In thisembodiment, the RF antenna 110 would be replaced by a photocell thatgenerates sufficient write voltage to exceed the threshold voltage. Forthe addressing commands, the RF transmitter 80 is replaced by a lightsource, and the commands may be transmitted digitally by pulsing theoptical transmitter, which can be a laser, flash lamp or other highintensity light source. It is noted that the light intensity must besufficient to generate adequate voltage, either singly or in conjunctionwith a second power generating device, in the photocell to write intomemory, but not so high that it damages the metal interconnect on thechip. With digital data transmission analog-to-digital anddigital-to-analog conversion circuitry can be eliminated.

The operation of programming the memory to record the process steps towhich the linked or adjacent matrix particle or support and linked orproximate molecule or biological particle is exposed involves placingthe memory device reasonably close [a distance on the order of about 1inch [25.4 mm] is presently contemplated, but longer distances should bepossible and shorter distances are also contemplated [suitable distancescan be determined empirically] to RF transmitter 80. The RF transmitter80 emits a carrier wave modulated by a signal generated by host computer122 using conventional RF technology. The carrier wave itself canprovide the power to the generate the programming voltage and theoperating voltage for the various devices via the rectifier, while themodulation signal provides the address instructions. As statedpreviously, since the memory only has to be “tagged” to record theexposure of the proximate or linked molecule or biological particle to agiven process, the address signal only has to carry information to turnon a single memory location, while the host computer 122 stores intomemory 120 the information linking the process information with thesingle memory location that was “tagged” to record exposure to theprocess step. Referring to FIG. 1, in which chemical building blocks A,C, and E are added to a molecule linked to a matrix with memory, and toFIG. 6, an illustrative example of how information is written onto aparticle is provided in Table 1. TABLE 1 X-REGISTER Y-REGISTER PROCESSSTEP ADDRESS ADDRESS A 1 8 C 2 4 E 3 2

For the step in which A is added, the address signal would increment thex-register 124 one location and increment the y-register 126 eightlocations, and then apply the programming voltage. The activation ofthis switch is indicated by an “A” at the selected address, although theactual value stored will be a binary “1”, indicating ON. [As described,for example, in U.S. Pat. No. 4,424,579; the manner in which theprogramming voltage is applied depends on whether the decoders havedepletion or enhancement transistors.] The host computer 122 would writeinto its memory 120 that for process A, the x-,y-address is 1,8. Uponremoval of the RF signal after recording process A, the voltage isremoved and the registers would reset to 0. For the step in which C isadded, the address signal would increment the x-register 124 twolocations and the y-register 126 four locations, then apply theprogramming voltage, as indicated by the letter “C”. The host computer120 would similarly record in memory that an indication of exposure toprocess C would be found at x-,y-address 2,4. Again, upon removal of theRF signal, the registers reset to 0 so that when the matrix particle'smemory is again exposed to RF following addition of block E, theregisters increment 3 and 2 locations, respectively, and the programmingvoltage is applied to turn on the switch, indicated by “E”. Desirablyall processing steps are automated.

After processing is completed, to read the information that has beenrecorded in the memory of the data storage unit, the host computer 122will inquire into the identity of the particle by generating a commandsignal to the registers to select the appropriate address locations todetermine whether the switch is on or off. If the switch is on, i.e., avoltage drop occurs at that point, the computer will create a recordthat the particle received a particular process step. Alternatively, thehost computer can generate an inquiry signal to sequentially look at allmemory locations to determine which switches have been turned on,recording all locations at which voltage drops occurred. The computerwill then compare the “on” locations to the process steps stored in itsmemory to identify the steps through which the subject particle wasprocessed.

If desired, individual particles can be identified by reserving certainmemory locations for identification only, for example, the first tworows of the x-register. In this case, particles will be passedseparately through the RF signal while the x-register is incremented toturn on switches at address locations 0,0, 1,0, 2,0, etc. Withindividual identification, the host computer 122 can first generate asignal to query a matrix particle memory to determine its identity, thenwrite the information with regard to the process performed, saving theprocess and particle information in the host computer memory 120.

Ideally, the tagging of particles which are exposed to a particularprocess would be performed in the process vessel containing all of theparticles. The presence, however, of a large number of particles mayresult in interference or result in an inability to generate asufficiently high voltage for programming all of the particlessimultaneously. This might be remedied by providing an exposure ofprolonged duration, e.g., several minutes, while stirring the vesselcontents to provide the greatest opportunity for all particles toreceive exposure to the RF signal. On the other hand, since eachparticle will need to be read individually, a mechanism for separatingthe particles may be used in both write and read operations. Also, ininstances in which each particle will have a different moleculeattached, each particle memory must be addressed separately.

An apparatus for separating the particles to allow individual exposureto the RF signal is illustrated in FIG. 7. Here, the particles areplaced in a vessel 140 which has a funnel 142, or other constrictedsection, which permits only one particle 150 to pass at a time. It isnoted that the particles, as illustrated, are, for purposes ofexemplification, depicted as spherical. The particles, however, can beof any shape, including asymmetric shapes. Where the particles areasymmetric or of other shapes, the size of the funnel exit and tubeshould be selected to fit the largest diameter of the particles closely.If a particular orientation of the particle is desired or required foreffective transmission, the tube and funnel exit should be designed andoriented to permit only particles in the proper alignment with the tubeto exit.

The RF transmitter 80 is positioned adjacent a tube 144 which receivesinput from funnel 142. When a particle passes through tube 144 the RFtransmitter provides a signal to write to or read from the particle'smemory. Means for initiating the RF transmission may include connectionto a mechanical gate or shutter 145 in the funnel 142 which controls theadmission of the particle into the tube. As illustrated in FIG. 7,however, optical means for detecting the presence of the matrix particlewith memory to initiate RF transmission are provided in the form of alaser 146 directed toward the tube 144, which is transparent to thewavelength of the light emitted by the laser. When the laser lightimpinges upon the particle [shown with dashed lines] it is reflectedtoward an optical detector 148 which provides a signal to the hostcomputer 122 to initiate the RF transmission. Alternatively, magneticmeans, or any other means for detecting the presence of the particle inthe tube 144 may be used, with the limitation that any electromagneticradiation used does not induce any reactions in the substances on theparticle's surface. After exposure of the individual particle to the RFsignal, the particle may be received in one or more vessels for furtherprocessing. As illustrated, tube 144 has an exemplary three-way splitterand selection means, shown here in dashed lines as mechanical gates, fordirecting the particles to the desired destination.

It is understood that the above description of operation and use of thedata storage devices, may be adapted for use with devices that containvolatile memories, such as EEPROMs, flash memory and DRAMs.

Other Memory or Encoded Devices Memory Devices

In addition to antifuse memory devices, other types ofelectrically-programmable read-only memories, preferably non-volatilememories, which are known in the art, may be used [see, e.g. U.S. Pat.No. 5,335,219]. Chips, such as those sold by Actel, Mosaic, LatticeSemiconductor, AVID, Anicare, Destron, Rayethon, Altera, ICT, Xilinix,Intel and Signetics [see, e.g., U.S. Pat. Nos. 4,652,528, 5,044,623,5,099,226, 5,218,343, 5,323,704, 4,333,072, 4,321,069, 4,318,658,5,121,748, 5,214,409, 5,235,326, 5,257,011 and 5,266,926] may be usedherein. Preprogrammed remotely addressable identification tags, such asthose used for tracking objects or animals [see, e.g., U.S. Pat. Nos.5,257,011, 5,235,326, 5,226,926, 5,214,409, 4,333,072, available fromAVID, Norco, Calif.; see, also U.S. Pat. Nos. 5,218,189, 5,416,486,4,952,928, 5,359,250] and remotely writable versions thereof are alsocontemplated for use herein. Preprogrammed tags may be used inembodiments, such as those in which tracking of linked molecules isdesired.

Alternatively, the matrices or strips attached thereto may be encodedwith a pre-programmed identifying bar code, such as an optical bar codethat will be encoded on the matrix and read by laser. Such precodeddevices may be used in embodiments in which parameters, such as locationin an automated synthesizer, are monitored. The identity of a product orreactant determined by its location or path, which is monitored byreading the chip in each device and storing such information in a remotecomputer. Read/write tags such as the IPTT-100 [BioMedic Data Systems,Inc., Maywood, N.J.; see, also U.S. Pat. Nos. 5,422,636, 5,420,579,5,262,772, 5,252,962, 5,250,962, and U.S. application Ser. No.08/322,644] are also contemplated for use herein.

Among the particularly preferred devices are the chips [particularly,the IPTT-100, Bio Medic Data Systems, Inc., Maywood, N.J.; see, alsoU.S. Pat. Nos. 5,422,636, 5,420,579, 5,262,772, 5,252,962 and 5,250,962and U.S. application Ser. No. 08/322,644,] that can be remotely encodedand remotely read. These devices, such as the IPTT-100 transponders thatare about 8 mm long, include a recording device, an EEPROM, a passivetransponder for receiving an input signal and transmitting an outputsignal in response. In some embodiments here, the devices are modifiedfor use herein by altering the geometry. They are folded in half and theantenna wrapped around the resulting folded structure. This permitsconvenient insertion into the microvessels and formation of othercombinations.

These devices include a power antenna means [see, e.g., U.S. Pat. No.5,250,944 and U.S. Pat. No. 5,420,579] for receiving the input signal,frequency generator and modulator means for receiving the input signalthe receive antenna means and for generating the output signal. Theoutput signal has a frequency different from the input frequency,outputs the output signal in response the input signal. The input signalhaving a first frequency, the output signal has a second frequency thatis a multiple of the first frequency, and is greater that the firstfrequency. It also includes a transmitting antenna means for receivingthe output signal from the frequency generator and modulator means andthat transmit the output signal. Data are stored within the transponderwithin a reprogrammable memory circuit that is programmed by the user[see, e.g., U.S. Pat. No. 5,422,636 and EP 0 526 173 A3]. A transponderscanner for scanning and programming the transponder is also available[Bio Medic Data Systems Inc. DAS-5001 CONSOLE™ System, e.g., U.S. Pat.No. 5,252,962 and U.S. Pat. No. 5,262,772].

Another such device is a 4 mm chip with an onboard antenna and an EEPROM[Dimensional Technology International, Germany]. This device can also bewritten to and read from remotely.

Also, ID tags available from IDTAG™ Inc, particularly the IDT150read/write transponder [ITDAG™ Ltd. Bracknell, Berks RG12 3XQ, UK],discussed above, are also preferred herein.

Encoded Devices

It is also contemplated herein, that the memory is not proximate to thematrix, but is separate, such as a remote computer or other recordingdevice. In these embodiments, the matrices are marked with a unique codeor mark of any sort. The identity of each mark is saved in the remotememory, and then, each time something is done to a molecule orbiological particle linked to each matrix, the information regardingsuch event is recorded and associated with the coded identity. Aftercompletion of, for example, a synthetic protocol, each matrix isexamined or read to identify the code. Retrieving information that fromthe remote memory that is stored with the identifying code will permitidentification or retrieval of any other saved information regarding thematrix.

For example, simple codes, including bar codes, alphanumeric charactersor other visually or identifiable codes or marks on matrices are alsocontemplated for use herein. When bar codes or other precoded devicesare used, the information can be written to an associated but remotememory, such as a computer or even a piece of paper. The computer storesthe bar code that a identifies a matrix particle or other code andinformation relating to the molecule or biological particle linked tothe matrix or other relevant information regarding the linked materialsor synthesis or assay. Instead of writing to an on-board memory,information is encoded in a remote memory that stores informationregarding the precoded identity of each matrix with bar code and linkedmolecules or biological particles. Thus, the precoded information isassociated with, for example, the identity of the linked molecule or acomponent thereof, or a position (such as X-Y coordinates in a grid).This information is transmitted to a memory for later retrieval. Eachtreatment or synthetic step that is performed on the linked molecule orbiological particle is transmitted to the remote memory and associatedwith the precoded ID.

For example, an amino acid is linked to a matrix particle that isencoded with or marked with a bar code or even a letter such as “A” orother coded mark. The identity the amino acid linked to the matrixparticle “A” is recorded into a memory. This particle is mixed withother particles, each with a unique identifier or mark, and this mixtureis then treated to a synthetic step. Each particle is individuallyscanned or viewed to see what mark is on each particle and the remotememory is written to describe the synthetic step, which is thenassociated with each unique identifier in the memory, such as thecomputer or piece of paper. Thus, in the remote memory the originalamino acid linked to particle A is stored. After the synthetic step, theidentify of the next amino acid is stored in the memory associated with“A” as is the identity of the next amino acid added. At the end of thesynthesis, the history of each particle can be read by scanning theparticle or visually looking at the particle and noting its bar code ormark, such as A. The remote memory is then queried to determine whatamino acids are linked to the particle identified as “A” [see, e.g.,FIG. 20].

For example, many combinatorial libraries contain a relatively smallnumber of discrete compounds [10²-10⁴] in a conveniently manipulablequantity, rather than millions of members in minute quantities. Thesesmall libraries are ideal for use with the methods and matrices withmemories herein. They may also be used in methods in which the memory isnot in proximity to the matrix, but is a remote memory, such as acomputer or a table of information stored even on paper. The systemdepicted in FIG. 20 is ideal for use in these methods.

Polypropylene or other inert polymer, including fluoropolymers orscintillating polymers are molded into a convenient geometry and size,such an approximately 5 mm×5 mm×5 mm cube [or smaller or larger] with aunique identifying code imprinted, preferably permanently, on one sideof each cube. If, for example, a three element code is used, based onall digits (0 to 9) and all letters of the alphabet, a collection of46,666 unique three element codes are available for imprinting on thecubes.

The cubes are surface grafted with a selected monomer [or mixture ofmonomer], such as styrene. Functionalization of the resulting polymerprovides a relatively large surface area for chemical syntheses andsubsequent assaying [on a single platform]. For example, a 5×5×5 mm³cube has a surface area of 150 mm², which is equivalent to about 2-5μmol achievable loading, which is about 1-2.5 mg of compounds with amolecular weight of about 500. A simple computer program or protocol candirect split and pool during synthesis and the information regardingeach building block of the linked molecules on each cube convenientlyrecorded in the memory [i.e., computer] at each step in the synthesis.

Since the cubes [herein called MACROCUBES™ or MACROBEADS™] arerelatively large, they can be read by the eye or any suitable deviceduring synthesis and the associated data can be manually entered into acomputer or even written down. The cubes can include scintillant orfluorophore or label and used in any of the assay formats describedherein or otherwise known to those of skill in the art.

For example, with reference to FIG. 20, polypropylene, polyethylene orfluophore raw material [any such material described herein, particularlythe Moplen resin e.g., V29G PP resin from Montell, Newark Del., adistributor for Himont, Italy] 1 is molded, preferably into a cube,preferably about 5×5×5 mm³ and engraved, using any suitable imprintingmethod, with a code, preferably a three element alphanumeric code, onone side. The cube can be weighted or molded so that it all cubes willorient in the same direction. The engraved cubes 2 are thensurface-grafted 3 and functionalized using methods described herein orknown to those of skill in this art, to produce cubes [MACROBEADS™ orMACROCUBES™] or devices any selected geometry 4.

Optically or Magnetically Programmed Devices

In addition to electrically-programmable means for storing informationon the matrix particles, optical or magnetic means may be used. Oneexample of an optical storage means is provided in U.S. Pat. No.5,136,572, issued Aug. 4, 1992, of Bradley, which is incorporated hereinby reference. Here, an array of stabilized diode lasers emits fixedwavelengths, each laser emitting light at a different wavelength.Alternatively, a tunable diode laser or a tunable dye laser, each ofwhich is capable of emitting light across a relatively wide band ofwavelengths, may be used. The recording medium is photochemically activeso that exposure to laser light of the appropriate wavelength will formspectral holes.

As illustrated In FIG. 8, an optical write/read system is configuredsimilar to that of the embodiment of FIG. 7, with a vessel 212containing a number of the particles which are separated and oriented bypassing through a constricted outlet into a write/read path 206 that hasan optically-transparent tube [i.e., optically transparent to therequired wavelength(s)] with a cross-section which orients the particlesas required to expose the memory surface to the laser 200 which iscapable of emitting a plurality of discrete, stable wavelengths. Gatingand detection similar to that described for the previous embodiment maybe used and are not shown. Computer 202 controls the tuning of laser 200so that it emits light at a unique wavelength to record a data point.Memory within computer 202 stores a record indicating which process stepcorresponds to which wavelength. For example, for process A, wavelengthλ₁, e.g., 630 nm [red], for process C, λ₂, e.g., 550 nm [yellow], andfor process E, λ₃, e.g., 480 nm [blue], etc. The recording medium 204 isconfigured to permit orientation to repeatably expose the recording sideof the medium to the laser beam each time it passes through tube 206.One possible configuration, as illustrated here, is a disc.

To write onto the recording medium 204, the laser 200 emits light of theselected wavelength to form a spectral hole in the medium. The light isfocussed by lens 208 to illuminate a spot on recording medium 204. Thelaser power must be sufficient to form the spectral hole. For reading,the same wavelength is selected at a lower power. Only this wavelengthwill pass through the spectral hole, where it is detected by detector210, which provides a signal to computer 202 indicative of the recordedwavelength. Because different wavelengths are used, multiple spectralholes can be superimposed so that the recording medium can be very smallfor purposes of tagging. To provide an analogy to the electrical memoryembodiments, each different wavelength of light corresponds to anaddress, so that each laser writes one bit of data. If a large number ofdifferent steps are to performed for which each requires a unique datapoint, the recording media will need to be sufficiently sensitive, andthe lasers well-stabilized, to vary only within a narrow band to assurethat each bit recorded in the media is distinguishable. Since only asingle bit of information is required to tag the particle at any givenstep, the creation of a single spectral hole at a specific wavelength iscapable of providing all of the information needed. The host computerthen makes a record associating the process performed with a particularlaser wavelength.

For reading, the same wavelength laser that was used to create thespectral hole will be the only light transmitted through the hole. Sincethe spectral holes cannot be altered except by a laser having sufficientpower to create additional holes, this type of memory is effectivelynonvolatile. Further, the recording medium itself does not have anyoperations occurring within its structure, as is the case in electricalmemories, so its structure is quite simple. Since the recording mediumis photochemically active, it must be well encased within an opticallytransmissive [to the active optical wavelength(s)], inert material toprevent reaction with the various processing substances while stillpermitting the laser light to impinge upon the medium. In many cases,the photochemical recording media may be erased by exposure to broadspectrum light, allowing the memory to be reused.

Writing techniques can also include the formation of pits in the medium.To read these pits, the detector 210 with be positioned on the same sideof the write/read tube 206 as the laser 200 to detect light reflectedback from the medium. Other types of optical data storage and recordingmedia may be used as are known in the art. For example, optical discs,which are typically plastic-encapsulated metals, such as aluminum, maybe miniaturized, and written to and read from using conventional opticaldisc technology. In such a system, the miniature discs must be alignedin a planar fashion to permit writing and reading. A modification of thefunnel system, described above, will include a flattened tube to insurethe proper orientation. Alternatively, the discs can be magneticallyoriented. Other optical recording media that may be appropriate for usein the recording devices and combinations herein include, but are notlimited to, magneto-optical materials, which provide the advantage oferasability, photochromic materials, photoferroelectric materials,photoconductive electro-optic materials, all of which utilize polarizedlight for writing and/or reading, as is known in the art. When using anyform of optical recording, however, considerations must be made toinsure that the selected wavelength of light will not affect orinterfere with reactions of the molecules or biological particles linkedto or in proximity to matrix particles.

Three Dimensional Optical Memories

Another device that is suitable for use in the matrix with memorycombinations are optical memories that employ rhodopsins, particularlybacteriorhodopsin [BR], or other photochromic substances that changebetween two light absorbing states in response to light of each of twowavelengths [see, e.g., U.S. Pat. Nos. 5,346,789, 5,253,198 and5,228,001; see, also Birge (1990) Ann. Rev. Phys. Chem. 41:683-733].These substances, particularly BR, exhibit useful photochromic andoptoelectrical properties. BR, for example, has extremely large opticalnonlinearities, and is capable of producing photoinduced electricalsignals whose polarity depends on the prior exposure of the material tolight of various wavelengths as well as on the wavelength of the lightused to induce the signal. There properties are useful for informationstorage and computation. Numerous applications of this material havebeen designed, including its use as an ultrafast photosignal detector,its use for dynamic holographic recording, and its use for data storage,which is of interest herein.

The rhodopsins include the visual rhodopsins, which are responsible forthe conversion of light into nerve impulses in the image resolving eyesof mollusks, anthropods, and vertebrates, and also bacteriorhodopsin[BR]. These proteins also include a class of proteins that servephotosynthetic and phototactic functions. The best known BR is the onlyprotein found in nature in a crystalline membrane, called the “purplemembrane” of Halobacterium Halobium. This membrane converts light intoenergy via photon-activated transmembrane proton pumping. Upon theabsorption of light, the BR molecule undergoes several structuraltransformations in a well-defined photocycle in which energy is storedin a proton gradient formed upon absorption of light energy. This protongradient is subsequently utilized to synthesize energy-rich ATP.

The structural changes that occur in the process of light-induced protonpumping of BR are reflected in alterations of the absorption spectra ofthe molecule. These changes are cyclic, and under usual physiologicalconditions bring the molecule back to its initial BR state after theabsorption of light in about 10 milliseconds. In less than a picosecondafter BR absorbs a photon, the BR produces an intermediate, known as the“J” state, which has a red-shifted absorption maximum. This is the onlylight-driven event in the photocycle; the rest of the steps arethermally driven processes that occur naturally. The first form, orstate, following the photon-induced step is called “K”, which representsthe first form of light-activated BR that can be stabilized by reducingthe temperature to 90° K. This form occurs about 3 picoseconds after theJ intermediate at room temperature. Two microseconds later there occursan “L” intermediate state which is, in turn, followed in 50 microsecondsby an “M” intermediate state.

There are two important properties associated with all of theintermediate states of this material. The first is their ability to bephotochemically converted back to the basic BR state. Under conditionswhere a particular intermediate is made stable, illumination with lightat a wavelength corresponding to the absorption of the intermediatestate in question results in regeneration of the BR state. In addition,the BR state and intermediates exhibit large two-photon absorptionprocesses which can be used to induce interconversions among differentstates.

The second important property is light-induced vectorial chargetransport within the molecule. In an oriented BR film, such a chargetransport can be detected as an electric signal. The electrical polarityof the signal depends on the physical orientation of molecules withinthe material as well as on the photochemical reaction induced. Thelatter effect is due to the dependence of charge transport direction onwhich intermediates [including the BR state] are involved in thephotochemical reaction of interest. For example, the polarity of anelectrical signal associated with one BR photochemical reaction isopposite to that associated with a second BR photochemical reaction. Thelatter reaction can be induced by light with a wavelength around 412 nmand is completed in 200 ns.

In addition to the large quantum yields and distinct absorptions of BRand M, the BR molecule [and purple membrane] has several intrinsicproperties of importance in optics. First, this molecule exhibits alarge two-photon absorption cross section. Second, the crystallinenature and adaptation to high salt environments makes the purplemembrane very resistant to degeneration by environmental perturbationsand thus, unlike other biological materials, it does not require specialstorage. Dry films of purple membrane have been stored for several yearswithout degradation. Furthermore, the molecule is very resistant tophotochemical degradation.

Thus, numerous optical devices, including recording devices have beendesigned that use BR or other rhodopsin as the recording medium [see,e.g., U.S. Pat. Nos. 5,346,789, 5,253,198 and 5,228,001; see, also Birge(1990) Ann. Rev. Phys. Chem. 41:683-733]. Such recording devices may beemployed in the methods and combinations provided herein.

Event-Detecting Embodiment

Another embodiment of the combinations herein utilizes a recordingdevice that can detect the occurrence of a reaction or event or thestatus of any external parameter, such as pH or temperature, and recorda such occurrence or parameter in the memory. Any of the above devicesmay be modified to permit such detection. For example, the chip with theantifuse memory array with decoder, rectifier components and RF antenna,can be modified by addition of a photodetector and accompanyingamplifier components as shown in FIG. 9. The photodetector will beselected so that it is sensitive to the frequencies of expectedphotoemissions from reactions of interest. To maintain the chip'spassive operation, the photodetector circuitry may use voltage suppliedby the same RF signal that is used to write other data to memory, sothat no detection of photoemission will occur unless RF or other poweris applied to provide bias and drain voltage. If an active device isused, the power supplied by the battery can provide operational voltageto the photodetector circuitry, independent of any transmitted signal.The voltage supplied by the photodetector can be used in a number ofdifferent ways. For example:

1) The threshold voltage for writing to memory will exceed the voltagesupplied by the RF signal, which will still contain the addressinformation. In order to write, additional voltage must be provided bythe photodetector so that the sum of the voltages exceeds the threshold.(V_(RF)<V_(T)<V_(RF)+V_(PD)). This permits the RF supplied voltage to goto the correct address, however, no writing will occur unless aphotoemission has been detected by the detector. Therefore, there willbe no record of exposure to a particular process step unless asufficient reaction has occurred to generate the required photoemission.Since the address signal can still get to the memory array without theextra voltage, reading of recorded data can be achieved without anyspecial circuitry. If the memory device is an active device, a similarmechanism can be used in which only the sum of the voltages issufficient to record an occurrence.

2) The threshold voltage for writing to memory will be provided by theRF signal alone, and the RF signal will include address information.(V_(T)<V_(RF)). However, unless voltage from the photodetector issupplied to a “gating” transistor, access to the memory array isprevented so that no writing occurs unless a photoemission is detected.(This embodiment is illustrated). This will require a special provisionfor opening the gate during read operations to permit access to thememory array. Since the gating transistor will conduct a signal only inthe event of photoemission, this embodiment will work equally well withpassive and active memory devices.

3) The RF signal provides sufficient voltage to exceed the thresholdvoltage. (V_(T)<V_(RF)). Voltage from the photodetector is used tocreate a write potential difference at an additional address locationwhich is carried in the RF signal. For example, if the RF signal isaddressing column 3, row 3, column 32 could be connected only to thephotodetector circuit's output so that, when a photoemission occurs, thewrite signal will create antifuses [or in the case of EEPROM, standardfuses] at addresses 3,3 and 32,3. If no photoemission occurs, onlyaddress 3,3 will have an antifuse formed, providing a record of exposureof the matrix to a particular process step even without the occurrenceof a detectable reaction. Special provisions, such as software withinthe host computer in combination with mask-programmed interconnectionswithin the decode circuitry of the memory device, must be made to assurethat more than one column in a single row of the array is polled duringread operations so that both memory locations are read.

In addition to the above-described methods for recording the occurrenceof photo-emitting reactions, the photodetector, while still integratedon the same substrate with the basic memory matrix for recordingtransmitted signals, can be connected to its own independent memorymatrix. In this embodiment, the photodetector's memory matrix can beconnected to separate transceiver circuitry with an antenna tuned to adifferent frequency from that of the basic memory. During the readoperation, the memory device will be exposed to two different radiofrequency signals, one for the basic memory, the other for thephotodetection circuit memory. If only the photoemission information isrequired, only the corresponding frequency signal need be providedduring the read operation.

Depending on the type of energy release that occurs during a reaction,other types of sensors may be used in addition to photodetectors or inplace thereof. In addition changes in ion concentration may also bedetected. Many such sensors will be capable of generating an electricalsignal that can be used as described above for the photodetectors. Thesesensing devices may also be incorporated onto the substrate andelectrically connected to the memory device, providing data pointswithin the device's memory under the appropriate write conditions. Forexample, temperature sensing elements can be made from semiconductorliquid crystal and fluorescent crystals, and addition to conventionalthermocouples created by placing two different metals in contact at thedetection point. It is also possible to include radiation, pH and pCO₂sensors in a similar manner, using materials that respond to thedetected variables by generating a voltage potential that can beconducted to the memory device and recorded.

The reaction-detecting embodiment may be advantageously used in assays,such as the SPA, HTRF, FET, FRET and FP assays described below. In theseassays, reaction, such as receptor binding, produces a detectablesignal, such as light, in the matrix. If a matrix with memory with aphotodetection circuit is used, occurrence of the binding reaction willbe recorded in memory.

C. The Combinations and Preparation Thereof

Combinations of a miniature recording device that contains or is a datastorage unit linked to or in proximity with matrices or supports used inchemical and biotechnical applications, such as combinatorial chemistry,peptide synthesis, nucleic acid synthesis, nucleic acid amplificationmethods, organic template chemistry, nucleic acid sequencing, screeningfor drugs, particularly high throughput screening, phage displayscreening, cell sorting, drug delivery, tracking of biological particlesand other such methods, are provided. These combinations of matrixmaterial with data storage unit [or recording device including the unit]are herein referred to as matrices with memories. These combinationshave a multiplicity of applications, including combinatorial chemistry,isolation and purification of target macromolecules, capture anddetection of macromolecules for analytical purposes, high throughputscreening protocols, selective removal of contaminants, enzymaticcatalysis, drug delivery, chemical modification, scintillation proximityassays, FET, FRET and HTRF assays, immunoassays, receptor bindingassays, drug screening assays, information collection and management andother uses. These combinations are particularly advantageous for use inmultianalyte analyses. These combinations may also be advantageouslyused in assays in which a electromagnetic signal is generated by thereactants or products in the assay. These combinations may be used inconjunction with or may include a sensor element, such as an elementthat measures a solution parameter, such as pH. Change in suchparameter, which is recorded in the memory will indicate a reactionevent of interest, such as induction of activity of a receptor or ionchannel, has occurred. The combination of matrix with memory is alsoadvantageously used in multiplex protocols, such as those in which amolecule is synthesized on the matrix, its identity recorded in thematrix, the resulting combination is used in an assay or in ahybridization reaction. Occurrence of the reaction can be detectedexternally, such as in a scintillation counter, or can be detected by asensor that writes to the memory in the matrix. Thus, combinations ofmatrix materials, memories, and linked or proximate molecules andbiological materials and assays using such combinations are provided.

The combinations contain (i) a miniature recording device that containsone or more programmable data storage devices [memories] that can beremotely read and in preferred embodiments also remotely programmed; and(ii) a matrix as described above, such as a particulate support used inchemical syntheses. The remote programming and reading is preferablyeffected using electromagnetic radiation, particularly radio frequencyor radar. Depending upon the application the combinations will includeadditional elements, such as scintillants, photodetectors, pH sensorsand/or other sensors, and other such elements.

1. Preparation of Matrix-Memory Combinations

In preferred embodiments, the recording device is cast in a selectedmatrix material during manufacture. Alternatively, the devices can bephysically inserted into the matrix material, the deformable gel-likematerials, or can be placed on the matrix material and attached by aconnector, such as a plastic or wax or other such material.Alternatively, the device or device(s) may be included in an inertcontainer in proximity to or in contact with matrix material.

2. Non-Linked Matrix-Memory Combinations

The recording device with memory can be placed onto the inner surface ofa vessel, such as a microtiter plate or vial or tube in which thereaction steps are conducted. Alternatively, the device can beincorporated into the vessel material, such into the a wall of eachmicrotiter well or vial or tube in which the reaction is conducted. Aslong as the molecules or biological particles remain associated with thewell, tube or vial, their identity can be tracked. Also of interestherein are the multiwell “chips” [such as those available from OrchidBiocomputer, Inc. Princeton, N.J., see, e.g., U.S. Pat. Nos. 5,047,371,4,952,531, 5,043,222, 5,277,724, 5,256,469 and Prabhu et al. (1992)Proc. SPIE-Int. Soc. Opt. Eng. 1847 NUMBER: Proceedings of the 1992International Symposium on Microelectronics, pp. 601-6], that aresilicone based chips that contain 10,000 microscopic wells connected byhair-thin glass tubes to tiny reservoirs containing reagents forsynthesis of compounds in each well. Each well can be marked with a codeand the code associated with the identity of the synthesized compound ineach well. Ultimately, a readable or read/write memory may beincorporated into each well, thus permitting rapid and readyidentification of the contents of each well.

In a particularly preferred embodiment, one or more recording deviceswith memory and matrix particles are sealed in a porous non-reactivematerial, such as polypropylene or teflon net, with a pore size smallerthan the particle size of the matrix and the device. Typically onedevice per about 1 to 50 mg, preferably 5 to 30, more preferably 5 to 20mg of matrix material, or in some embodiments up to gram, generally 50to 250 mg, preferably 150 mg to about 200 mg, and one device is sealedin a porous vessel a microvessel [MICROKAN™]. The amount of matrixmaterial is a function of the size of the device and the application inwhich the resulting matrix with memory is used, and, if necessary can beempirically determined. Generally, smaller sizes are desired, and theamount of material will depend upon the size of the selected recordingdevice.

The resulting microvessels are then encoded, reactions, such assynthetic reactions, performed, and read, and if desired used in desiredassays or other methods.

3. Preparation of Matrix-Memory-Molecule or Biological ParticleCombinations

In certain embodiments, combinations of matrices with memories andbiological particle combinations are prepared. For example, libraries[e.g., bacteria or bacteriophage, or other virus particles or otherparticles that contain genetic coding information or other information]can be prepared on the matrices with memories, and stored as such forfuture use or antibodies can be linked to the matrices with memories andstored for future use.

4. Combinations for Use in Proximity Assays

In other embodiments the memory or recording device is coated orencapsulated in a medium, such as a gel, that contains one or morefluophors or one or more scintillants, such as 2,5-diphenyloxazole [PPO]and/or 1,4-bis-[5-phenyl-(oxazolyl)]benzene [POPOP] or FlexiScint [a gelwith scintillant available from Packard, Meriden, Conn.] or yttriumsilicates. Any fluophore or scintillant or scintillation cocktail knownto those of skill in the art may be used. The gel coated or encaseddevice is then coated with a matrix suitable, such as glass orpolystyrene, for the intended application or application(s). Theresulting device is particularly suitable for use as a matrix forsynthesis of libraries and subsequent use thereof in scintillationproximity assays.

Similar combinations in non-radioactive energy transfer proximityassays, such as HTRF, FP, FET and FRET assays, which are describedbelow. These luminescence assays are based on energy transfer between adonor luminescent label, such as a rare earth metal cryptate [e.g., Eutrisbipyridine diamine (EuTBP) or Tb tribipyridine diamine (TbTBP)] andan acceptor luminescent label, such as, when the donor is EuTBP,allopycocyanin (APC), allophycocyanin B, phycocyanin C or phycocyanin R,and when the donor is TbTBP, a rhodamine, thiomine, phycocyanin R,phycoerythrocyanin, phycoerythrin C, phycoerythrin B or phycoerythrin R.Instead of including a scintillant in the combination, a suitablefluorescent material, such as allopycocyanin (APC), allophycocyanin B,phycocyanin C, phycocyanin R; rhodamine, thiomine, phycocyanin R,phycoerythrocyanin, phycoerythrin C, phycoerythrin B or phycoerythrin Ris included. Alternatively, a fluorescent material, such a europiumcryptate is incorporated in the combination.

5. Other Variations and Embodiments

The combination of memory with matrix particle may be further linked,such as by welding using a laser or heat, to an inert carrier or othersupport, such as a teflon strip. This strip, which can be of anyconvenient size, such as 1 to 10 mm by about 10 to 100 μM will renderthe combination easy to use and manipulate. For example, these memorieswith strips can be introduced into 10 cm culture dishes and used inassays, such as immunoassays, or they can be used to introduce bacteriaor phage into cultures and used in selection assays. The strip may beencoded or impregnated with a bar code to further provide identifyinginformation.

Microplates containing a recording device in one or a plurality of wellsare provided. The plates may further contain embedded scintillant or acoating of scintillant [such as FlashPlate™, available from DuPont NEN®,and plates available from Packard, Meriden, Conn.] FLASHPLATE™ is a 96well microplate that is precoated with plastic scintillant for detectionof β-emitting isotopes, such as ¹²⁵I, ³H, ³⁵S, ¹⁴C and ³³P. A moleculeis immobilized or synthesized in each well of the plate, each memory isprogrammed with the identify of each molecule in each well. Theimmobilized molecule on the surface of the well captures a radiolabeledligand in solution results in detection of the bound radioactivity.These plates can be used for a variety of radioimmmunoassays [RIAs],radioreceptor assays [RRAs], nucleic acid/protein binding assays,enzymatic assays and cell-based assays, in which cells are grown on theplates.

Another embodiment is depicted in FIG. 19. The reactive sites, such asamines, on a support matrix [1 in the FIGURE] in combination with amemory [a MICROKAN™, a MICROTUBE™, a MACROBEAD™, a MICROCUBE™ or othermatrix with memory combination] are differentiated by reacting them witha selected reaction of Fmoc-glycine and Boc-glycine, thereby producing adifferentiated support [2]. The Boc groups gropus on 2 are thendeprotected with a suitable agent such as TFA, to produce 3. Theresulting fee amine groups are coupled with a fluophore for mixture Aand B, to produce a fluorescent support 4, which can be used insubsequent syntheses or for linkage of desired molecules or biologicalparticles, and then used in fluorescence assays and SPAs.

D. The Recording and Reading and Systems

Systems for recording and reading information are provided. The systemsinclude a host computer or decoder/encoder instrument, a transmitter, areceiver and the data storage device. The systems also can include afunnel-like device or the like for use in separating and/or taggingsingle memory devices. In practice, an EM signal, preferably a radiofrequency signal is transmitted to the data storage device. The antennaor other receiver means in the device detects the signal and transmitsit to the memory, whereby the data are written to the memory and storedin a memory location.

Mixtures of the matrix with memory-linked molecules or biologicalparticles may be exposed to the EM signal, or each matrix with memory[either before, after or during linkage of the biological particles ormolecules] may be individually exposed, using a device, such as thatdepicted herein, to the EM signal. Each matrix with memory, as discussedbelow, will be linked to a plurality of molecules or biologicalparticles, which may be identical or substantially identical or amixture of molecules or biological particles depending, upon theapplication and protocol in which the matrix with memory and linked [orproximate] molecules or biological particles is used. The memory can beprogrammed with data regarding such parameters.

The location of the data, which when read and transmitted to the hostcomputer or decoder/encoder instrument, corresponds to identifyinginformation about linked or proximate molecules or biological particles.The host computer or decoder/encoder instrument can either identify thelocation of the data for interpretation by a human or another computeror the host computer or the decoder/encoder can be programmed with a keyto interpret or decode the data and thereby identify the linked moleculeor biological particle.

As discussed above, the presently preferred system for use is theIPTT-100 transponder and DAS-5001 CONSOLE™ [Bio Medic Data Systems,Inc., Maywood, N.J.; see, e.g., U.S. Pat. Nos. 5,422,636, 5,420,579,5,262,772, 5,252,962 and 5,250,962, 5,252,962 and 5,262,772].

These systems may be automated or may be manual.

Manual System

The presently preferred manual system includes a transponder,particularly the BMDS transponder described below or an IDTAG™transponder, described above, and uses the corresponding reading andwriting device, which has been reconfigured and repackaged, such as inFIG. 17, described in the EXAMPLES An example of the operation of thesystem of FIG. 17 is illustrated in FIG. 18 and described in EXAMPLE 4.Briefly, the user manually places a microvessel 180 within the recessedarea 176 so that the interrogation signal 185 provides a response to thecontrollers indicating the presence on the microvessel, and informationis read from or written to the transponder.

This will include microvessels, such as MICROKANS™ or MICROTUBES™,read/writer hardware [such as that available from BMDS or IDTAG™]connected to a PC and software running on the PC that performs a userinterface and system control function. The software is designed tofacilitate the a number of aspects of synthetic combinatorial chemistrylibraries, including: organization, planning and design, synthesiscompound formula determination, molecular weight computation, reportingof plans, status and results.

In particular, for each chemical library, the software creates a database file. This file contains all of the information pertinent to thelibrary, including chemical building blocks to be used, the design ofthe library in terms of steps and splits, and what synthesis has beenperformed. This file oriented approach allows many different chemicallibrary projects to be conducted simultaneously. The software allows theuser to specify what chemical building blocks are to be used and theirmolecular weights. The user specifies the number of steps, the number of“splits” at each step, and what chemical building blocks are to be usedat each split. The user may also enter the name of the pharmacophore andits molecular weight. Additionally, the user may specify graphicalchemical diagrams for the building blocks and the pharmacophore. Thisinformation is useful in displaying resulting compounds. The softwarerecords all of the above “design” information. It computes and displaysthe size of the library. It may also predict the range of molecularweights of the resulting compounds.

For example, the user specifies that there will be eight chemicalbuilding blocks. Their names are entered, and the user enters a uniqueletter codes for each: A, B, C, D, E, F, G and H. The user specifiesthat there will be three steps. Step one will have four splits,appending the A, B, C and D building blocks. Step two will also havefour splits, adding the B, D, E and H building blocks. Step three willhave six splits, adding the B, C, D, E, F and G building blocks. Thesoftware computes that the library will contain 96 (4×6×5=96) uniquecompounds. With the planning and design completed, the software helpsthe user perform the synthesis steps. This is done in concert with thereader/writer hardware [transceiver or a scanner, such as the BMDS-DAS5003] or a similar device available form IDTAG Ltd [Bracknell, BerksRG12 3XQ, UK] and devices, such as the MICROKAN™ or MICROTUBE™microvessel with memory devices. Before the synthesis begins, themicrovessels are filled with polymer resin. The microvessel devices are,one at a time placed upon the scanner. The device and software reads thecontents of the data encoded in the recording device, transponder, suchas the BMDS tag or the IDTAG™ tag, contained in each microvessel. Thesoftware, chooses which building block shall be added to the compoundcontained in each microvessel. It directs the transceiver to writeencoded data to the transponder, indicating which building block thisis. The software displays a message which directs the user to place themicrovessel in the appropriate reaction vessel so that the chosenbuilding block will be added. This process is repeated a plurality oftimes with each microvessel and for each synthetic step the plannedsteps of the library.

The software then uses the scanner to read a tag and receive its encodedinformation. Using the user-entered compound names stored in thelibrary's data base, the software translates the encoded informationinto the names of the chemical building blocks. The software can alsodisplay compounds graphically, using the graphical information specifiedby the user. The software calculates the molecular weight of compoundsfrom the data provided for the pharmacophore and building blocks. Thesoftware facilitates the recording of progress through the aboveprocess. The software generates displays and reports which illustratethis and all of the above planning, design, compound data, and graphicalrepresentations of compounds.

E. Tools and Applications Using Matrices with Memories

1. Tools

The matrix with memory and associated system as described herein is thebasic tool that can be used in a multitude of applications, includingany reaction that incorporates a functionally specific (i.e. in thereaction) interaction, such as receptor binding. This tool is thencombined with existing technologies or can be modified to produceadditional tools.

For example, the matrix with memory combination, can be designed as asingle analyte test or as a multianalyte test and also as a multiplexedassay that is readily automated. The ability to add one or a mixture ofmatrices with memories, each with linked or proximate molecule orbiological particle to a sample, provides that ability to simultaneouslydetermine multiple analytes and to also avoid multiple pipetting steps.The ability to add a matrix with memory and linked molecules orparticles with additional reagents, such as scintillants, provides theability to multiplex assays.

As discussed herein, in one preferred embodiment the matrices areparticulate and include adsorbed, absorbed, or otherwise linked orproximate, molecules, such as peptides or oligonucleotides, orbiological particles, such as cells. Assays using such particulatememories with matrices may be conduced “on bead” or “off bead”. On beadassays are suitable for multianalyte assays in which mixtures ofmatrices with linked molecules are used and screened against a labeledknown. Off bead assays may also be performed; in these instances theidentity of the linked molecule or biological particle must be knownprior to cleavage or the molecule or biological particle must be in somemanner associated with the memory.

In other embodiments the matrices with memories use matrices that arecontinuous, such as microplates, and include a plurality of memories,preferably one memory/well. Of particular interest herein are matrices,such as Flash Plates™ [NEN, Dupont], that are coated or impregnated withscintillant or fluophore or other luminescent moiety or combinationthereof, modified by including a memory in each well. The resultingmemory with matrix is herein referred to as a luminescing matrix withmemory. Other formats of interest that can be modified by including amemory in a matrix include the Multiscreen Assay System [Millipore] andgel permeation technology.

2. Scintillation Proximity Assays (SPAs) and Scintillant-ContainingMatrices with Memories

Scintillation proximity assays are well known in the art [see, e.g.,U.S. Pat. No. 4,271,139; U.S. Pat. No. 4,382,074; U.S. Pat. No.4,687,636; U.S. Pat. No. 4,568,649; U.S. Pat. No. 4,388,296; U.S. Pat.No. 5,246,869; International PCT Application No. WO 94/26413;International PCT Application No. WO 90/03844; European PatentApplication No. 0 556 005 A1; European Patent Application No. 0 301 769A1; Hart et al. (1979) Molec. Immunol. 16:265-267; Udenfriend et al.(1985) Proc. Natl. Acad. Sci. U.S.A. 82:8672-8676; Nelson et al. (1987)Analyt. Biochem 165:287-293; Heath, et al. (1991) Methodol. Surv.Biochem. Anal. 21:193-194; Mattingly et al. (1995) J. Memb. Sci.98:275-280; Pernelle (1993) Biochemistry 32:11682-116878; Bosworth etal. (1989) Nature 341:167-168; and Hart et al. (1989) Nature 341:265].Beads [particles] and other formats, such as plates and membranes havebeen developed.

SPA assays refer to homogeneous assays in which quantifiable lightenergy produced and is related to the amount of radioactively labelledproducts in the medium. The light is produced by a scintillant that isincorporated or impregnated or otherwise a part of a support matrix. Thesupport matrix is coated with a receptor, ligand or other capturemolecule that can specifically bind to a radiolabeled analyte, such as aligand.

a. Matrices

Typically, SPA uses fluomicrospheres, such as diphenyloxazole-latex,polyacrylamide-containing a fluophore, and polyvinyltoluene [PVT]plastic scintillator beads, and they are prepared for use by adsorbingcompounds into the matrix. Also fluomicrospheres based on organicphosphors have been developed. Microplates made from scintillationplastic, such as PVT, have also been used [see, e.g., International PCTApplication No. WO 90/03844]. Numerous other formats are presentlyavailable, and any format may be modified for use herein by includingone or more recording devices.

Typically the fluomicrospheres or plates are coated with acceptormolecules, such as receptors or antibodies to which ligand bindsselectively and reversibly. Initially these assays were performed usingglass beads containing fluors and functionalized with recognition groupsfor binding specific ligands [or receptors], such as organic molecules,proteins, antibodies, and other such molecules. Generally the supportbodies used in these assays are prepared by forming a porous amorphousmicroscopic particle, referred to as a bead [see, e.g., European PatentApplication No. 0 154,734 and International PCT Application No. WO91/08489]. The bead is formed from a matrix material such as acrylamide,acrylic acid, polymers of styrene, agar, agarose, polystyrene, and othersuch materials, such as those set forth above. Cyanogen bromide has beenincorporated into the bead into to provide moieties for linkage ofcapture molecules or biological particles to the surface. Scintillantmaterial is impregnated or incorporated into the bead by precipitationor other suitable method. Alternatively, the matrices are formed fromscintillating material [see, e.g., International PCT Application No. WO91/08489, which is based on U.S. application Serial No. 07/444,297; see,also U.S. Pat. No. 5,198,670], such as yttrium silicates and otherglasses, which when activated or doped respond as scintillators. Dopantsinclude Mn, Cu, Pb, Sn, Au, Ag, Sm, and Ce. These materials can beformed into particles or into continuous matrices. For purposes herein,the are used to coat, encase or otherwise be in contact with one or aplurality of recording devices.

Assays are conducted in normal assay buffers and requires the use of aligand labelled with an isotope, such as ³H and ¹²⁵I, that emitslow-energy radiation that is readily dissipated easily an aqueousmedium. Because ³H β particles and ¹²⁵I Auger electrons have averageenergies of 6 and 35 keV, respectively, their energies are absorbed bythe aqueous solutions within very small distances (˜4 μm for ³H βparticles and 35 μm for ¹²⁵I Auger electrons). Thus, in a typicalreaction of 0.1 ml to 0.4 ml the majority of unbound labelled ligandswill be too far from the fluomicrosphere to activate the fluor. Boundligands, however, will be in sufficiently close proximity to thefluomicrospheres to allow the emitted energy to activate the fluor andproduce light. As a result bound ligands produce light, but free ligandsdo not. Thus, assay beads emit light when they are exposed to theradioactive energy from the label bound to the beads through theantigen-antibody linkage, but the unreacted radioactive species insolution is too far from the bead to elicit light. The light from thebeads will be measured in a liquid scintillation counter and will be ameasure of the bound label.

Memories with matrices for use in scintillation proximity assays [SPA]are prepared by associating a memory with a matrix that includes ascintillant. In the most simple embodiment, matrix particles withscintillant [fluomicrospheres] are purchased from Amersham, Packard, NETechnologies [(formerly Nuclear Enterprises, Inc.) San Carlos, Calif.]or other such source and are associated with a memory, such as byincluding one or more of such beads in a MICROKAN™ microvessel with arecording device. Typically, such beads as purchased are derivatized andcoated with selected moieties, such as streptavidin, protein A, biotin,wheat germ agglutinin [WGA], and polylysine. Also available areinorganic fluomicrospheres based on cerium-doped yttrium silicate orpolyvinyltoluene (PVT). These contain scintillant and may be coated andderivatized.

Alternatively, small particles of PVT impregnated with scintillant areused to coat recording devices, such as the IPTT-100 devices [Bio MedicData Systems, Inc., Maywood, N.J.; see, also U.S. Pat. Nos. 5,422,636,5,420,579, 5,262,772, 5,252,962, 5,250,962, 5,074,318, and RE 34,936]that have been coated with a protective material, such as polystyrene,teflon, a ceramic or anything that does not interfere with the readingand writing EM frequency(ies). Such PVT particles may be manufactured orpurchased from commercial sources such as NE TECHNOLOGY, INC. [catalog#191A, 1-10 μm particles]. These particles are mixed with agarose oracrylamide, styrene, vinyl or other suitable monomer that willpolymerize or gel to form a layer of this material, which is coated onpolystyrene or other protective layer on the recording device. Thethickness of the layers may be empirically determined, but they must besufficiently thin for the scintillant to detect proximate radiolabels.To make the resulting particles resistant to chemical reaction they maybe coated with polymers such as polyvinyltoluene or polystyrene, whichcan then be further derivatized for linkage and/or synthesis ofmolecules and biological particles. The resulting beads are hereincalled luminescening matrices with memories, and when used in SPAformats are herein referred to as scintillating matrices with memories.

The scintillating matrices with memories beads can be formed bymanufacturing a bead containing a recording device and includingscintillant, such as 2,5-diphenyloxazole [PPO] and/or1,4-bis-[5-phenyl-(oxazolyl)]benzene [POPOP] as a coating. Theseparticles or beads are then coated with derivatized polyvinyl benzene orother suitable matrix on which organic synthesis, protein synthesis orother synthesis can be performed or to which organic molecules,proteins, nucleic acids, biological particles or other such materialscan be attached. Attachment may be effected using any of the methodsknown to those of skill in the art, including methods described herein,and include covalent, noncovalent, direct and indirect linkages.

Molecules, such as ligands or receptors or biological particles arecovalently coupled thereto, and their identity is recorded in thememory. Alternatively, molecules, such as small organics, peptides andoligonucleoties, are synthesized on the beads as described herein sothat history of synthesis and/or identity of the linked molecule isrecorded in the memory. The resulting matrices with memory particleswith linked molecules or biological particles may be used in anyapplication in which SPA is appropriate. Such applications, include, butare not limited to: radioimmunoassays, receptor binding assays, enzymeassays and cell biochemistry assays.

For use herein, the beads, plates and membranes are either combined witha recording device or a plurality of devices, or the materials used inpreparing the beads, plates or membranes is used to coat, encase orcontact a recording device. Thus, microvessels [MICROKANS™] containingSPA beads coated with a molecule or biological particle of interest;microplates impregnated with or coated with scintillant, and recordingdevices otherwise coated with, impregnated with or contacted withscintillant are provided.

To increase photon yield and remove the possibility of loss of fluor,derivatized fluomicrospheres based on yttrium silicate, that is dopedselectively with rare earth elements to facilitate production of lightwith optimum emission characteristics for photomultipliers andelectronic circuitry have been developed [see, e.g., European PatentApplication No. 0 378 059 B1; U.S. Pat. No. 5,246,869]. In practice,solid scintillant fibers, such as cerium-loaded glass or based on rareearths, such as yttrium silicate, are formed into a matrix. The glassesmay also include activators, such as terbium, europium or lithium.Alternatively, the fiber matrix may be made from a scintillant loadedpolymer, such as polyvinyltoluene. Molecules and biological particlescan be adsorbed to the resulting matrix.

For use herein, these fibers may be combined in a microvessel with arecording device [i.e., to form a MICROKAN™]. Alternatively, the fibersare used to coat a recording device or to coat or form a microplatecontaining recording devices in each well. The resulting combinationsare used as supports for synthesis of molecules or for linkingbiological particles or molecules. The identity and/or location and/orother information about the particles is encoded in the memory and theresulting combinations are used in scintillation proximity assays.

Scintillation plates [e.g., FlashPlates™, NEN Dupont, and other suchplates] and membranes have also been developed [see, Mattingly et al.(1995) J. Memb. Sci. 98:275-280] that may be modified by including amemory for use as described herein. The membranes, which can containpolysulfone resin M.W. 752 kD, polyvinylpyrrolidone MW 40 kDA,sulfonated polysulfone, fluor, such as p-bis-o-methylstyrylbenzene, POPand POPOP, may be prepared as described by Mattingly, but used to coat,encase or contact a recording device. Thus, instead of applying thepolymer solution to a glass plate the polymer solution is applied to therecording device, which, if need is pre-coated with a protectivecoating, such as a glass, teflon or other such coating.

Further, as shown in the Examples, the recording device may be coatedwith glass, etched and the coated with a layer of scintillant. Thescintillant may be formed from a polymer, such as polyacrylamide,gelatin, agarose or other suitable material, containing fluophors, ascintillation cocktail, FlexiScint [Packard Instrument Co., Inc.,Downers Grove, Ill.] NE Technology beads [see, e.g., U.S. Pat. No.4,588,698 for a description of the preparation of such mixtures].Alternatively, microplates that contain recording devices in one or morewells may be coated with or impregnated with a scintillant ormicroplates containing scintillant plastic may be manufactured withrecording devices in each well. If necessary, the resulting bead,particle or continuous matrix, such as a microplate, may be coated witha thin layer polystyrene, teflon or other suitable material. In allembodiments it is critical that the scintillant be in sufficientproximity to the linked molecule or biological particle to detectproximate radioactivity upon interaction of labeled molecules or labeledparticles with the linked molecule or biological particle.

The resulting scintillating matrices may be used in any application forwhich scintillation proximity assays are used. These include, ligandidentification, single assays, multianalyte assays, includingmulti-ligand and multi-receptor assays, radioimmunoassays [RIAs], enzymeassays, and cell biochemistry assays [see, e.g., International PCTApplication No. WO 93/19175, U.S. Pat. No. 5,430,150, Whitford et al.(1991) Phytochemical Analysis 2:134-136; Fenwick et al. (1994) Anal.Proc. Including Anal. Commun. 31:103-106; Skinner et al. (1994) Anal.Biochem. 223:259-265; Matsumura et al. (1992) Life Sciences51:1603-1611; Cook et al. (1991) Structure and Function of the AsparticProteinases, Dunn, ed., Penum Press, NY, pp. 525-528; Bazendale et al.in (1990) Advances in Prostaglandin, Thromboxane and LeukotrieneResearch, Vol. 21, Samuelsson et al., eds., Raven Press, NY, pp302-306].

b. Assays

(1) Receptor Binding Assays

Scintillating matrices with memories beads can be used, for example, inassays screening test compounds as agonists or antagonists of receptorsor ion channels or other such cell surface protein. Test compounds ofinterest are synthesized on the beads or linked thereto, the identity ofthe linked compounds is encoded in the memory either during or followingsynthesis, linkage or coating. The scintillating matrices with memoriesare then incubated with radiolabeled [¹²⁵I, ³H, or other suitableradiolabel] receptor of interest and counted in a liquid scintillationcounter. When radiolabeled receptor binds to any of the structuressynthesized or linked to the bead, the radioisotope is in sufficientproximity to the bead to stimulate the scintillant to emit light. Incontrast By contrast, if a receptor does not bind, less or noradioactivity is associated with the bead, and consequently less lightis emitted. Thus, at equilibrium, the presence of molecules that areable to bind the receptor may be detected. When the reading iscompleted, the memory in each bead that emits light [or more light thana control] queried and the host computer, decoder/encoder, or scannercan interpret the memory in the bead and identify the active ligand.

(a) Multi-Ligand Assay

Mixtures of scintillating matrices with memories with a variety oflinked ligands, which were synthesized on the matrices or linked theretoand their identities encoded in each memory, are incubated with a singlereceptor. The memory in each light-emitting scintillating matrix withmemory is queried and the identity of the binding ligand is determined.

(b) Multi-Receptor Assays

Similar to conventional indirect or competitive receptor binding assaysthat are based on the competition between unlabelled ligand and a fixedquantity of radiolabeled ligand for a limited number of binding sites,the scintillating matrices with memories permit the simultaneousscreening of a number of ligands for a number of receptor subtypes.

Mixtures of receptor coated beads [one receptor type/per bead; eachmemory encoded with the identity of the linked receptor] are reactedwith labeled ligands specific for each receptor. After the reaction hasreached equilibrium, all beads that emit light are reacted with a testcompound. Beads that no longer emit light are read.

For example receptor isoforms, such as retinoic acid receptor isoforms,are each linked to a different batch of scintillating matrix with memorybeads, and the identity of each isoform is encoded in the memories oflinked matrices. After addition of the radiolabeled ligand(s), such as³H-retinoic acid, a sample of test compounds [natural, synthetic,combinatorial, etc.] is added to the reaction mixture, mixed andincubated for sufficient time to allow the reaction to reachequilibrium. The radiolabeled ligand binds to its receptor, which hasbeen covalently linked to the bead and which the emitted short rangeelectrons will excite the fluophor or scintillant in the beads,producing light. When unlabelled ligand from test mixture is added, ifit displaces the labeled ligand it will diminish or stop the fluorescentlight signal. At the end of incubation period, the tube can be measuredin a liquid scintillation counter to demonstrate if any of the testmaterial reacted with receptor family. Positive samples [reduced or nofluorescence] will be further analyzed for receptor subtyping byquerying their memories with the RF detector. In preferred embodiments,each bead will be read and with a fluorescence detector and RF scanner.Those that have a reduced fluorescent signal will be identified and thelinked receptor determined by the results from querying the memory.

The same concept can be used to screen for ligands for a number ofreceptors. In one example. FGF receptor, EGF receptor, and PDGF receptorare each covalently linked to a different batch of scintillating matrixwith memory beads. The identity of each receptor is encoded in eachmemory. After addition of the ¹²⁵I-ligands [¹²⁵I-FGF, ¹²⁵I-EGF, and¹²⁵I-PDGF] a sample of test compounds [natural, synthetic,combinatorial, etc.) is added to the tube containing¹²⁵I-ligand-receptor-beads, m mixed and incubated for sufficient time toallow the reaction to reach equilibrium. The radiolabeled ligands bindto their respective receptors receptor that been covalently linked tothe bead. By virtue of proximity of the label to the bead, the emittedshort range electrons will excite the fluophor in the beads. Whenunlabelled ligand from test mixture is added, if it displaces the any ofthe labeled ligand it will diminish or stop the fluorescent signal. Atthe end of incubation period, the tube can be measured in a liquidscintillation counter to demonstrate if any of the test material reactedwith the selected receptor family. Positive samples will be furtheranalyzed for receptor type by passing the resulting complexes measuringthe fluorescence of each bead and querying the memories by exposing themto RF or the selected EM radiation. The specificity of test ligand isdetermined by identifying beads with reduced fluorescence that anddetermining the identity of the linked receptor by querying the memory.

(c) Other Formats

Microspheres, generally polystyrene typically about 0.3 μm-3.9 μm, aresynthesized with scintillant inside can either be purchased or preparedby covalently linking scintillant to the monomer prior to polymerizationof the polystyrene or other material. They can then be derivatized [orpurchased with chemical functional groups], such as —COOH, and —CH₂OH.Selected compounds or libraries are synthesized on the resultingmicrospheres linked via the functional groups, as described herein, orreceptor, such as radiolabeled receptor, can be coated on themicrosphere. The resulting “bead” with linked compounds, can used in avariety of SPA and related assays, including immunoassays, receptorbinding assays, protein:protein interaction assays, and other suchassays in which the ligands linked to the scintillant-containingmicrospheres are reacted with memories with matrices that are coatedwith a selected receptor.

For example, ¹²⁵I-labeled receptor is passively coated on the memorywith matrix and then mixed with ligand that is linked to a thescintillant-containing microspheres. Upon binding the radioisotope intois brought into close proximity to the scintillant in which effectiveenergy transfer from the β particle will occur, resulting in emission oflight.

Alternatively, the memory with matrix [containing scintillant] can alsobe coated with ³H-containing polyer on which the biological target[i.e., receptor, protein, antibody, antigen] can be linked [viaadsorption or via a functional group]. Binding of the ligand brings thescintillant into close proximity to the label, resulting in lightemission.

(2) Cell-based Assays

Cell-based assays, which are fundamental for understanding of thebiochemical events in cells, have been used with increasing frequency inbiology, pharmacology, toxicology, genetics, and oncology [see, e.g.,Benjamin et al. (1992) Mol. Cell. Biol. 12:2730-2738] Such cell linesmay be constructed or purchased [see, e.g., the Pro-Tox Kit availablefrom Xenometrix, Boulder Colo.; see, also International PCT ApplicationNo. WO 94/7208 cell lines]. Established cell lines, primary cellculture, reporter gene systems in recombinant cells, cells transfectedwith gene of interest, and recombinant mammalian cell lines have beenused to set up cell-based assays. For example Xenometrix, Inc. [Boulder,Colo.] provides kits for screening compounds for toxicological endpointsand metabolic profiles using bacteria and human cell lines. Screening iseffected by assessing activation of regulatory elements of stress genesfused to reporter genes in bacteria, human liver or colon cell lines andprovide information on the cytotoxicity and permeability of testcompounds.

In any drug discovery program, cell-based assays offer a broad range ofpotential targets as well as information on cytotoxicity andpermeability. The ability to test large numbers of compounds quickly andefficiently provides a competitive advantage in pharmaceutical leadidentification.

High throughput screening with cell-based assays is often limited by theneed to use separation, wash, and disruptive processes that compromisethe functional integrity of the cells and performance of the assay.Homogeneous or mix-and-measure type assays simplify investigation ofvarious biochemical events in whole cells and have been developed usingscintillation microplates [see, e.g., International PCT Application No.WO 94/26413, which describes scintillant plates that are adapted forattachment and/or growth of cells and proximity assays using suchcells]. In certain embodiment herein, cell lines such as those describedin International PCT Application No. WO 94/17208 are be plated onscintillant plates, and screened against compounds synthesized onmatrices with memories. Matrices with memories encoded with the identityof the linked molecule will be introduced into the plates, the linkagescleaved and the effects of the compounds assessed. Positive compoundswill be identified by querying the associated memory.

The scintillant base plate is preferably optically transparent toselected wavelengths that allow cells in culture to be viewed using aninverted phase contrast microscope, and permit the material to transmitlight at a given wavelength with maximum efficiency. In addition thebase retains its optical properties even after exposure to incident betaradiation from radioisotopes as well as under stringent radiationconditions required for sterilization of the plates. The base plate canbe composed of any such optically transparent material containingscintillant, e.g., a scintillant glass based on lanthanide metalcompounds. Typically, the base plate is composed of any plasticmaterial, generally formed from monomer units that include phenyl ornaphthyl moieties in order to absorb incident radiation energy fromradionuclides which are in close proximity with the surface. Preferablythe plastic base plate is composed of polystyrene or polyvinyltoluene,into which the scintillant is incorporated. The scintillant includes,but is not limited to: aromatic hydrocarbons such as p-terphenyl,p-quaterphenyl and their derivatives, as well as derivatives of theoxazoles and 1,3,4-oxadiazoles, such as2-(4-t-butylphenyl)-5-(4-biphenyl)-1,3,4-oxadiazole and2,5-diphenyloxazole. Also included in the polymeric composition may be awavelength shifter such as 1,4-bis(5-phenyl-2-oxazolyl)benzene,9,10-diphenylanthracene, 1,4-bis(2-methylstyryl)-benzene, and other suchcompounds. The function of the wavelength shifter is to absorb the lightemitted by the scintillant substance and re-emit longer wavelength lightwhich is a better match to the photo-sensitive detectors used inscintillation counters. Other scintillant substances and polymer bodiescontaining them are known to those of skill in this art [see, e.g.,European Patent Application No. 0 556 005 A1].

The scintillant substances can be incorporated into the plastic materialof the base by a variety of methods. For example, the scintillators maybe dissolved into the monomer mix prior to polymerization, so that theyare distributed evenly throughout the resultant polymer. Alternativelythe scintillant substances may be dissolved in a solution of the polymerand the solvent removed to leave a homogeneous mixture. The base plateof disc may be bonded to the main body of the well or array of wells,which itself may be composed of a plastic material includingpolystyrene, polyvinyltoluene, or other such polymers. In the case ofthe multi-well array, the body of the plate may be made opaque, i.e.,non-transparent and internally reflective, in order to completelyexclude transmission of light and hence minimize “cross-talk.” This isaccomplished by incorporating into the plastic at the polymerizationstage a white dye or pigment, for example, titanium dioxide. Bonding ofthe base plate to the main body of the device can be accomplished by anysuitable bonding technique, for example, heat welding, injection moldingor ultrasonic welding.

For example, a 96-well plate is constructed to the standard dimensionsof 96-well microtiter plates 12.8 cm×8.6 cm×1.45 cm with wells in anarray of 8 rows of 12 wells each. The main body of the plate isconstructed by injection molding of polystyrene containing a loading ofwhite titanium oxide pigment at 12%. At this stage, the wells of themicrotiter plate are cylindrical tubes with no closed end. A base plateis formed by injection molding of polystyrene containing2-(4-t-butylphenyl)-5-(4-biphenyl)-1,3,4-oxadiazole (2%) and9,10-diphenylanthracene (0.5%). The base plate has been silk screenprinted with a grid array to further reduce crosstalk. The base plate isthen fused in a separate operation to the body by ultrasonic welding,such that the grid array overlies the portions of the microtiter platebetween the wells.

A 24-well device is constructed to the dimensions 12.8×8.6×1.4 cm with24 wells in an array of 4 rows of 6 wells. The main body of the plate[not including the base of each well] is constructed by injectionmolding of polystyrene containing 12% white titanium oxide pigment. Thebase 24 of each well is injection molded with polystyrene containing2-(4-t-butylphenyl)-5-(4-biphenylyl)-1,3,4-oxadizaole [2%] and9,10-diphenylanthracene [0.5%]. The heat from the injected base plasticresults in fusion to the main body giving an optically transparent baseto the well.

The plates may contain multiple wells that are continuous or that areeach discontinuous from the other wells in the array, or they may besingle vessels that have, for example, an open top, side walls and anoptically transparent scintillant plastic base sealed around the loweredge of the side walls.

In another format the plate, is a single well or tube. The tube may beconstructed from a hollow cylinder made from optically transparentplastic material and a circular, scintillant containing, plastic disc.The two components are welded together so as to form a single well ortube suitable for growing cells in culture. As in the plate format,bonding of the circular base plate to the cylindrical portion isachieved by any conventional bonding technique, such as ultrasonicwelding. The single well or tube may be any convenient size, suitablefor scintillation counting. In use, the single well may either becounted as an insert in a scintillation vial, or alternatively as aninsert in a scintillation vial, or alternatively as an insert in amulti-well plate of a flat bed scintillation counter. In this lattercase, the main body of the multi-well plate would need to be opaque forreasons given earlier.

The various formats are selected according to use. They may be used forgrowing cells and studying cellular biochemical processes in livingcells or cell fragments. The 96-well plate is a standard format used inexperimental cell biology and one that is suitable for use in a flat bedscintillation counter [e.g., Wallac Microbeta or Packard Top Count]. Inthe multi-well format, it is an advantage to be able to prevent “crosstalk” between different wells of the plate that may be used formonitoring different biological processes using different amounts ortypes of radioisotope. Therefore the main body of the plate can be madefrom opaque plastic material. The 24-well plate format is commonly usedfor cell culture. This type of plate is also suitable for counting in aflat bed scintillation counter. The dimensions of the wells will belarger.

As an alternative format, the transparent, scintillant containingplastic disc is made to be of suitable dimensions so as to fit into thebottom of a counting vessel. The counting vessel is made fromnon-scintillant containing material such as glass or plastic and shouldbe sterile in order to allow cells to grow and the correspondingcellular metabolic processes to continue. Cells are first cultured onthe disc, which is then transferred to the counting vessel for thepurposes of monitoring cellular biochemical processes.

The culture of cells on the scintillation plastic base plate of thewells (or the disc) involves the use of standard cell cultureprocedures, e.g., cells are cultured in a sterile environment at 37° C.in an incubator containing a humidified 95% air/5% CO₂ atmosphere.Various cell culture media may be used including media containingundefined biological fluids such as fetal calf serum, or media which isfully defined and serum-free. For example, MCDB 153 is a selectivemedium for the culture of human keratinocytes [Tsao et al. (1982) J.Cell. Physiol. 110:219-229].

These plates are suitable for use with any adherent cell type that canbe cultured on standard tissue culture plasticware, including culture ofprimary cells, normal and transformed cells derived from recognizedsources species and tissue sources. In addition, cells that have beentransfected with the recombinant genes may also be cultured using theinvention. There are established protocols available for the culture ofmany of these diverse cell types [see, e.g., Freshney et al. (1987)Culture of Animal Cells: A Manual of Basic Technique, 2nd Edition, AlanR. Liss Inc.]. These protocols may require the use of specializedcoatings and selective media to enable cell growth and the expression ofspecialized cellular functions.

The scintillating base plate or disc, like all plastic tissue cultureware, requires surface modification in order to be adapted for theattachment and/or growth of cells. Treatment can involves the use ofhigh voltage plasma discharge, a well established method for creating anegatively charged plastic surface [see, e.g., Amstein et al. (1975) J.Clinical Microbiol. 2:46-54]. Cell attachment, growth and the expressionof specialized functions can be further improved by applying a range ofadditional coatings to the culture surface of the device. These caninclude: (i) positively or negatively charged chemical coatings such aspoly-lysine or other biopolymers [McKeehan et al. (1976) J. Cell Biol.71:727-734 (1976)]; (ii) components of the extracellular matrixincluding collagen, laminin, fibronectin [see, e.g., Kleinman et al.(1987) Anal. Biochem. 166:1-13]; and (iii) naturally secretedextracellular matrix laid down by cells cultured on the plastic surface[Freshney et al. et al. (1987) Culture of Animal Cells: A Manual ofBasic Technique, 2nd Edition, Alan R. Liss Inc.]. Furthermore, thescintillating base plate may be coated with agents, such as lectins, oradhesion molecules for attachment of cell membranes or cell types thatnormally grow in suspension. Methods for the coating of plasticware withsuch agents are known [see, e.g., Boldt et al. (1979) J. Immunol.123:808].

In addition, the surface of the scintillating layer may be coated withliving or dead cells, cellular material, or other coatings of biologicalrelevance. The interaction of radiolabeled living cells, or otherstructures with this layer can be monitored with time allowing processessuch as binding, movement to or from or through the layer to bemeasured.

Virtually all types of biological molecules can be studied. A anymolecule or complex of molecules that interact with the cell surface orthat can be taken up, transported and metabolized by the cells, can beexamined using real time analysis. Examples of biomolecules will includereceptor ligands, protein and lipid metabolite precursors (e.g., aminoacids, fatty acids), nucleosides and any molecule that can beradiolabeled. This would also include ions such as calcium, potassium,sodium and chloride, that are functionally important in cellularhomeostasis, and which exist as radioactive isotopes. Furthermore,viruses and bacteria and other cell types, which can be radiolabeled asintact moieties, can be examined for their interaction with monolayeradherent cells grown in the scintillant well format.

The type of radioactive isotope that can be used with this system willtypically include any of the group of isotopes that emit electronshaving a mean range up to 2000 μm in aqueous medium. These will includeisotopes commonly used in biochemistry such as [³H], [¹²⁵I], [¹⁴C],[³⁵S], [⁴⁵Ca], [³³p], and [³²p], but does not preclude the use of otherisotopes, such as [⁵⁵Fe], [¹⁰⁹Cd] and [⁵¹Cr] that also emit electronswithin this range. The wide utility of the invention for isotopes ofdifferent emission energy is due to the fact that the current formatsenvisaged would allow changes to the thickness of the layer containing ascintillant substance, thereby ensuring that all the electron energy isabsorbed by the scintillant substance. Furthermore, cross-talkcorrection software is available which can be utilized with all highenergy emitters. Applications using these plates include proteinsynthesis, Ca²⁺ transport, receptor-ligand binding, cell adhesion, sugartransport and metabolism, hormonal stimulation, growth factor regulationand stimulation of motility, thymidine transport, and protein synthesis.

For use in accord with the methods herein, the scintillant plates caninclude a memory in each well, or alternatively, memory withmatrix-linked compounds will be added to each well. The recording devicewith memory may be impregnated or encased or placed in wells of theplate, typically during manufacture. In preferred embodiments, however,the memories are added to the wells with adsorbed or linked molecules.

In one embodiment, matrices with memories with linked molecules areintroduced into scintillant plates in which cells have been cultured[see, e.g., International PCT Application No. WO 94/26413]. For example,cells will be plated on the transparent scintillant base 96-wellmicroplate that permits examination of cells in culture by invertedphase contrast microscope and permits the material to transmit light ata given wavelength with maximum efficiency. Matrices with memories towhich test compounds linked by preferably a photocleaveable linker areadded to the wells. The identity of each test compound is encoded in thememory of the matrix during synthesis if the compound is synthesized onthe matrix with memory or when the compound is linked to the matrix.

Following addition of matrix with memory to the well and release ofchemical entities synthesized on the beads by exposure to light or otherprocedures, the effects of the chemical released from the beads on theselected biochemical events, such as signal transduction, cellproliferation, protein or DNA synthesis, in the cells can be assessed.In this format receptor binding Such events include, but are not limitedto: whole cell receptor-ligand binding [agonist or antagonist],thymidine or uridine transport, protein synthesis (using, for example,labeled cysteine, methionine, leucine or proline], hormone and growthfactor induced stimulation and motility, and calcium uptake.

In another embodiment, the memories are included in the plates eitherplaced in the plates or manufactured in the wells of the plates. Inthese formats, the identities of the contents of the well is encodedinto the memory. Of course it is understood, that the informationencoded and selection of encased or added memories depends upon theselected protocol.

In another format, cells will be plated on the tissue culture plate,after transferring the matrices with memories and release of compoundssynthesized on the beads in the well. Cytostatic, cytotoxic andproliferative effects of the compounds will be measured usingcalorimetric [MTT, XTT, MTS, Alamar blue, and Sulforhodamine B],fluorimetric [carboxyfluorescein diacetate], or chemiluminescentreagents [i.e., CytoLite™, Packard Instruments, which is used in ahomogeneous luminescent assay for cell proliferation, cell toxicity andmulti-drug resistance].

For example, cells that have been stably or transiently transfected witha specific gene reporter construct containing an inducible promoteroperatively linked to a reporter gene that encodes an indicator proteincan be calorimetrically monitored for promoter induction. Cells will beplated on the tissue culture 96-well microtiter plate and after additionof memories with matrices in the wells and release of chemical entitiessynthesized on the matrices, the effect of the compound released fromthe beads on the gene expression will be assessed. The CytosensorMicrophysiometer [Molecular Devices] evaluates cellular responses thatare mediated by G protein-linked receptors, tyrosine kinase-linkedreceptors, and ligand-gated ion channels. It measures extracellular pHto assess profiles of compounds assessed for the ability to modulateactivities of any of the these cell surface proteins by detectingsecretion of acid metabolites as a result of altered metabolic states,particularly changes in metabolic rate. Receptor activation requires useof ATP and other energy resources of the cell thereby leading toincreased in cellular metabolic rate. For embodiments herein, thememories with matrices, particularly those modified for measuring pH,and including linked test compounds, can be used to track and identifythe added test compound added and also to detect changes in pH, therebyidentifying linked molecules that modulate receptor activities.

3. Memories with Matrices for Non-Radioactive Energy Transfer ProximityAssays

Non-radioactive energy transfer reactions, such as FET or FRET, FP andHTRF assays, are homogeneous luminescence assays based on energytransfer are carried out between a donor luminescent label and anacceptor label [see, e.g., Cardullo et al. (1988) Proc. Natl. Acad. Sci.U.S.A. 85:8790-8794; Peerce et al. (1986) Proc. Natl. Acad. Sci. U.S.A.83:8092-8096; U.S. Pat. No. 4,777,128; U.S. Pat. No. 5,162,508; U.S.Pat. No. 4,927,923; U.S. Pat. No. 5,279,943; and International PCTApplication No. WO 92/01225]. The donor label is usually a rare earthmetal cryptate, particularly europium trisbipyridine diamine [EuTBP] orterbium trisbipyridine diamine [TbTBP] and an acceptor luminescent,presently fluorescent, label. When the donor is EuTBP, the acceptor ispreferably allopycocyanin [APC], allophycocyanin B, phycocyanin C orphycocyanin R, and when the donor is TbTBP, the acceptor is a rhodamine,thiomine, phycocyanin R, phycoerythrocyanin, phycoerythrin C,phycoerythrin B or phycoerythrin R.

Energy transfer between such donors and acceptors is highly efficient,giving an amplified signal and thereby improving the precision andsensitivity of the assay. Within distances characteristic ofinteractions between biological molecules, the excitation of afluorescent label (donor) is transferred non radiatively to a secondfluorescent label (acceptor). When using europium cryptate as the donor,APC, a phycobiliprotein of 5 kDa, is presently the preferred acceptorbecause it has high molar absorptivity at the cryptate emissionwavelength providing a high transfer efficiency, emission in a spectralrange in which the cryptate signal is insignificant, emission that isnot quenched by presence of sera, and a high quantum yield. When usingEu³⁺ cryptate as donor, an amplification of emitted fluorescence isobtained by measuring APC emission.

The rare earth cryptates are formed by the inclusion of a luminescencelanthanide ion in the cavity of a macropolycyclic ligand containing2,2′-biphyridine groups as light absorbers [see, e.g., U.S. Pat. No.5,162,508; U.S. Pat. No. 4,927,923; U.S. Pat. No. 5,279,943; andInternational PCT Application No. WO 92/01225]. Preferably the Eu3⁺trisbypryidine diamine derivative, although the acceptor may be used asthe label, is cross-linked to antigens, antibodies, proteins, peptides,and oligonucleotides and other molecules of interest.

For use herein, matrices with memories are prepared that incorporateeither the donor or, preferably the acceptor, into or on the matrix. Inpractice, as with the scintillating matrices with memories, the matricesmay be of any format, i.e. particulate, or continuous, and used in anyassay described above for the scintillating matrices. For example, therecording device is coated with a protective coating, such as glass orpolystyrene. If glass it can be etched. As with preparation of thescintillating matrices with memories, compositions containing the donoror preferably acceptor, such as APC, and typically a polymer or gel, arecoated on the recording device or the device is mixed with thecomposition to produce a fluorescing matrix with memory. To make thesematrices resistant to chemical reaction, if needed, they may be coatedwith polymers such as polyvinylbenzene or polystyrene. Molecules, suchas the constituents of combinatorial libraries, are synthesized on thefluorescing matrices with memories, or molecules or biological particlesare linked thereto, the identity of the synthesized molecules or linkedmolecules or biological particles is encoded in memory, and theresulting matrices with memories employed in any suitable assay,including any of those described for the scintillating memories withmatrices. In particular, these homogeneous assays using long-livedfluorescence rare earth cryptates and amplification by non radiativeenergy transfer have been adapted to use in numerous assays includingassays employing ligand receptor interaction, signal transduction,transcription factors (protein-protein interaction), enzyme substrateassays and DNA hybridization and analysis [see, Nowak (1993) Science270:368; see, also, Velculescu et al. (1995) Science 270:484-487, andSchena et al. (1995) Science 270:467-470, which describe methodsquantitative and simultaneous analysis of a large number of transcriptsthat are particularly suited for modification using matrices withmemories]. Each of these assays may be modified using the fluorescingmatrices with memories provided herein.

For example, a receptor will be labeled with a europium cryptate [(wherethe matrices with memories incorporate, for example allophycocyanin(APC)] or will be labeled with APC, where the matrices incorporate aeuropium cryptate. After mixing receptor and mixtures of matrices withdifferent ligands, the mixture is exposed to laser excitation at 337 nm,and, if reaction has occurred, typical signals of europium cryptate andAPC over background are emitted. Measurement with an interference filtercentered at 665 nm selects the signal of the APC labeled receptor fromthat of europium cryptate labeled ligand on the beads. If particulate,the memories of matrices that emit at 665, can be queried to identifylinked ligands.

4. Other Applications Using Memories with Matrices and LuminescingMemories with Matrices

a. Natural Product Screening

In the past, the vast majority of mainline pharmaceuticals have beenisolated form natural products such as plants, bacteria, fungus, andmarine microorganisms. Natural products include microbials, botanicals,animal and marine products. Extracts of such sources are screened fordesired activities and products. Selected products include enzymes[e.g., hyaluronidase], industrial chemicals [e.g., petroleum emulsifyingagents], and antibiotics [e.g., penicillin]. It is generally consideredthat a wealth of new agents still exist within the natural productspool. Large mixtures of natural products, even within a fermentationbroth, can be screened using the matrices with memory combinationslinked, for example, to peptides, such as antigens or antibody fragmentsor receptors, of selected and known sequences or specificities, or toother biologically active compounds, such as neurotransmitters, cellsurface receptors, enzymes, or any other identified biological target ofinterest. Mixtures of these peptides linked to memory matrices can beintroduced into the natural product mixture. Individual bindingmatrices, detected by an indicator, such as a fluorometric dye, can beisolated and the memory queried to determine which linked molecule orbiological particle is bound to a natural product.

b. Immunoassays and Immunodiagnostics

The combinations and methods provided herein represent major advances inimmunodiagnotics. Immunoassays [such as ELISAs, RIAs and EIAs (enzymeimmunoassays)] are used to detect and quantify antigens or antibodies.

(1) Immunoassays

Immunoassays detect or quantify very small concentrations of analytes inbiological samples. Many immunoassays use solid supports in whichantigen or antibody is covalently, non-covalently, or otherwise, such asvia a linker, attached to a solid support matrix. The support-boundantigen or antibody is then used as an analyte in the assay. As withnucleic acid analysis, the resulting antibody-antigen complexes or othercomplexes, depending upon the format used, rely on radiolabels or enzymelabels to detect such complexes.

The use of antibodies to detect and/or quantitate reagents [“antigens”]in blood or other body fluids has been widely practiced for many years.Two methods have been most broadly adopted. The first such procedure isthe competitive binding assay, in which conditions of limiting antibodyare established such that only a fraction [usually 30-50%] of a labeled[e.g., radioisotope, fluophore or enzyme] antigen can bind to the amountof antibody in the assay medium. Under those conditions, the addition ofunlabeled antigen [e.g., in a serum sample to be tested] then competeswith the labeled antigen for the limiting antibody binding sites andreduces the amount of labeled antigen that can bind. The degree to whichthe labeled antigen is able to bind is inversely proportional to theamount of unlabeled antigen present. By separating the antibody-boundfrom the unbound labeled antigen and then determining the amount oflabeled reagent present, the amount of unlabeled antigen in the sample[e.g., serum] can be determined.

As an alternative to the competitive binding assay, in the labeledantibody; or “immunometric” assay [also known as “sandwich” assay], anantigen present in the assay fluid is specifically bound to a solidsubstrate and the amount of antigen bound is then detected by a labeledantibody [see, e.g., Miles et al. (1968) Nature 29:186-189; U.S. Pat.No. 3,867,517; U.S. Pat. No. 4,376,110]. Using monoclonal antibodiestwo-site immunometric assays are available [see, e.g., U.S. Pat. No.4,376,110]. The “sandwich” assay has been broadly adopted in clinicalmedicine. With increasing interest in “panels” of diagnostic tests, inwhich a number of different antigens in a fluid are measured, the needto carry out each immunoassay separately becomes a serious limitation ofcurrent quantitative assay technology.

Some semi-quantitative detection systems have been developed [see, e.g.,Buechler et al. (1992) Clin. Chem. 38:1678-1684; and U.S. Pat. No.5,089,391] for use with immunoassays, but no good technologies yet existto carefully quantitate a large number of analytes simultaneously [see,e.g., Ekins et al. (1990) J. Clin. Immunoassay 13:169-181] or to rapidlyand conveniently track, identify and quantitate detected analytes.

The methods and memories with matrices provided herein provide a meansto quantitate a large number of analytes simultaneously and to rapidlyand conveniently track, identify and quantitate detected analytes.

(2) Multianalyte Immunoassays

The combinations of matrix with memories provided herein permits thesimultaneous assay of large numbers of analytes in any format. Ingeneral, the sample that contains an analyte, such as a ligand or anysubstance of interest, to be detected or quantitated, is incubated withand bound to a protein, such as receptor or antibody, or nucleic acid orother molecule to which the analyte of interest binds. In oneembodiment, the protein or nucleic acid or other molecule to which theanalyte of interest binds has been linked to a matrix with memory priorto incubation; in another embodiment, complex of analyte or ligand andprotein, nucleic acid or other molecule to which the analyte of interestbinds is linked to the matrix with memory after the incubation; and in athird embodiment, incubation to form complexes and attachment of thecomplexes to the matrix with memory are simultaneous. In any embodiment,attachment is effected, for example, by direct covalent attachment, bykinetically inert attachment, by noncovalent linkage, or by indirectlinkage, such as through a second binding reaction [i.e., biotin-avidin,Protein A-antibody, antibody-hapten, hybridization to form nucleic acidduplexes of oligonucleotides, and other such reactions andinteractions]. The complexes are detected and quantitated on the solidphase by virtue of a label, such as radiolabel, fluorescent label,luminophore label, enzyme label or any other such label. The informationthat is encoded in the matrix with memory depends upon the selectedembodiment. If, for example, the target molecule, such as the protein orreceptor is bound to the solid phase, prior to complexation, theidentity of the receptor and/or source of the receptor may be encoded inthe memory in the matrix.

For example, the combinations provided herein are particularly suitablefor analyses of multianalytes in a fluid, and particularly formultianalyte immunoassays. In one example, monoclonal antibodies veryspecific for carcinoembryonic antigen [CEA], prostate specific antigen[PSA], CA-125, alphafetoprotein [AFP], TGF-β, IL-2, IL-8 and IL-10 areeach covalently attached to a different batch of matrices with memoriesusing well-established procedures and matrices for solid phase antibodyassays. Each antibody-matrix with memory complex is given a specificidentification tag, as described herein.

A sample of serum from a patient to be screened for the presence orconcentration of these antigens is added to a tube containing two ofeach antibody-matrix with memory complex [a total of 16 beads, orduplicates of each kind of bead]. A mixture of monoclonal antibodies,previously conjugated to fluorescent dyes, such as fluorescein orphenyl-EDTA-Eu chelate, reactive with different epitopes on each of theantigens is then added. The tubes are then sealed and the contents aremixed for sufficient time [typically one hour] to allow any antigenspresent to bind to their specific antibody-matrix with memory-antigencomplex to produce antibody-matrix with memory-antigen-labeled antibodycomplexes. At the end of the time period, these resulting complexes arebriefly rinsed and passed through an apparatus, such as that set forthin FIG. 7, but with an additional light source. As each complex passesthrough a light source, such as a laser emitting at the excitationwavelength of fluorescein, about 494 nm, or 340 nm for the Eu chelatecomplex, its fluorescence is measured and quantitated by reading theemitted photons at about 518 nm for fluorescein or 613 nm forphenyl-EDTA-Eu, and as its identity is determined by the specific signalreceived by the RF detector. In this manner, eight different antigensare simultaneously detected and quantitated in duplicate.

In another embodiment, the electromagnetically tagged matrices withrecorded information regarding linked antibodies can be used with othermultianalyte assays, such as those described by Ekins et al. [(1990) J.Clin. Immunoassay 13:169-181; see, also International PCT ApplicationsNos. 89/01157 and 93/08472, and U.S. Pat. Nos. 4,745,072, 5,171,695 and5,304,498]. These methods rely on the use of small concentrations ofsensor-antibodies within a few μm² area. Individual memories withmatrices, or an array of memories embedded in a matrix are used.Different antibodies are linked to each memory, which is programmed torecord the identity of the linked antibody. Alternatively, the antibodycan be linked, and its identity or binding sites identified, and theinformation recorded in the memory. Linkage of the antibodies can beeffected by any method known to those of skill in this art, but ispreferably effected using cobalt-iminodiacetate coated memories [see,Hale (1995) Analytical Biochem. 231:46-49, which describes means forimmobilization of antibodies to cobalt-iminodiacetate resin] mediatedlinkage particularly advantageous. Antibodies that are revesibly boundto a cobalt-iminodiacetate resin are attached in exchange insert mannerwhen the cobalt is oxidized from the +2 to +3 state. In this state theantibodies are not removed by metal chelating regents, high salt,detergents or chaotropic agents. They are only removed by reducingagents. In addition, since the metal binding site in antibodies is inthe C-terminus heavy chain, antibodies so-bound are oriented with thecombining site directed away from the resin.

In particular antibodies are linked to the matrices with memories. Thematrices are either in particular form or in the form of a slab with anarray of recording devices linked to the matrices or microtiter dish orthe like with a recording device in each well. Antibodies are thenlinked either to each matrix particle or to discrete “microspots” on theslab or in the microtiter wells. In one application, prior to use ofthese matrices with memories, they are bound to a relatively lowaffinity anti-idiotype antibody [or other species that specificallyrecognizes the antibody binding site, such as a single chain antibody orpeptidomimetic] labeled with a fluophore [e.g., Texas Red, see, Ekins etal. (1990) J. Clin. Immunoassay 13:169-181] to measure the concentrationof and number of available binding sites present on each matrix withmemory particle or each microspot, which information is then encodedinto each memory for each microspot or each particle. These low affinityantibodies are then eluted, and the matrices can be dried and storeduntil used.

Alternatively or additionally, the memories in the particles or at eachmicrospot could be programmed with the identity or specificity of thelinked antibody, so that after reaction with the test sample andidentification of complexed antibodies, the presence and concentrationof particular analytes in the sample can be determined. They can be usedfor multianalyte analyses as described above.

After reaction with the test sample, the matrices with memories arereacted with a second antibody, preferably, although not necessarily,labeled with a different label, such as a different fluophore, such asfluorescein. After this incubation, the microspots or each matrixparticle is read by passing the particle through a laser scanner [suchas a confocal microscope, see, e.g., Ekins et al. (1990) J. Clin.Immunoassay 13:169-181; see also U.S. Pat. No. 5,342,633] to determinethe fluorescence intensity. The memories at each spot or linked to eachparticle are queried to determine the total number of available bindingsites, thereby permitting calculation of the ratio of occupied tounoccupied binding sites.

Equilibrium dialysis and modifications thereof has been used to studythe interaction of antibody or receptor or other protein or nucleic acidwith low molecular weight dialyzable molecules that bind to the antibodyor receptor or other protein or nucleic acid. For applications herein,the antibody, receptor, protein or nucleic acid is linked to solidsupport (matrix with memory) and is incubated with the ligand.

In particular, this method may be used for analysis of multiple bindingagents [receptors], linked to matrices with memories, that compete foravailable ligand, which is present in limiting concentration. Afterreaction, the matrices with memories linked to the binding agents[receptors] with the greatest amount of bound ligand, are the bindingagents [receptors] that have the greatest affinity for the ligand.

The use of matrices with memories also permits simultaneousdetermination of K_(a) values of multiple binding agents [receptors] orhave multiple ligands. For example, a low concentration of labeledligand is mixed with a batch of different antibodies bound to matriceswith memories. The mixture is flowed through a reader [i.e., a Coultercounter or other such instrument that reads RF and the label] couldsimultaneously measure the ligand [by virtue of the label] and identityof each linked binding agent [or linked ligand] as the chip is read.After the reaction equilibrium [determined by monitoring progress of thereaction] labeled ligand is added and the process of reading label andthe chips repeated. This process is repeated until all binding sites onthe binding agent [or ligand] approach saturation, thereby permittingcalculation of K_(a) values and binding sites that were available.

c. Selection of Antibodies and Other Screening Methods

(1) Antibody Selection

In hybridoma preparation and selection, fused cells are plated into, forexample, microtiter wells with the matrices with memory-tagged antibodybinding reagent [such as protein A or Co-chelate [see, e.g., Smith etal. (1992) Methods: A Companion to Methods in Enzymology 4, 73 (1992);Ill et al. (1993) Biophys J. 64:919: Loetscher et al. (1992) J.Chromatography 595:113-199; U.S. Pat. No. 5,443,816; Hale (1995)Analytical Biochem. 231:46-49]. The solid phase is removed, pooled andprocessed batchwise to identify the cells that produce antibodies thatare the greatest binders [see, e.g., U.S. Pat. No. 5,324,633 for methodsand device for measuring the binding affinity of a receptor to a ligand;or the above method by which phage libraries are screened for highestK^(A) phage, it, limiting labeled antigen].

(2) Antibody Panning

Memories with matrices with antibody attached thereto [e.g. particularlyembodiments in which the matrix is a plate] may be used in antibodypanning [see, e.g., Wysocki et al. (1978) Proc. Natl. Acad. Sci. U.S.A.75:2844-48; Basch et al. (1983) J. Immunol. Methods 56:269; Thiele etal. (1986) J. Immunol. 136:1038-1048; Mage et al. (1981) Eur. J.Immunol. 11:226; Mage et al. (1977) J. Immunol. Methods 15:47-56; see,also, U.S. Pat. Nos. 5,217,870 and 5,087,570, for descriptions of thepanning method]. Antibody panning was developed as a means tofractionate lymphocytes on the basis of surface phenotype based on theability of antibody molecules to adsorb onto polystyrene surfaces andretain the ability to bind antigen. Originally [Wysocki et al. (1978)Proc. Natl. Acad. Sci. U.S.A. 75:2844-2848] polystyrene dishes coatedwith antibodies specific for cell surface antigens and permit cells tobind to the dishes, thereby fractionating cells. In embodiments herein,polystyrene or other suitable matrix is associated with a memory deviceand coated with an antibody, whose identity is recorded in the memory.Mixtures of these antibody coated memories with matrices can be mixedwith cells, and multiple cell types can be sorted and identified byquerying the memories to which cells have bound.

d. Phage Display

Phage, viruses, bacteria and other such manipulable hosts and vectors[referred to as biological particles] can be modified to expressselected antigens [peptides or polypeptides] on their surfaces by, forexample, inserting DNA encoding the antigen into the host or vectorgenome, at a site such as in the DNA encoding the coat protein, suchthat upon expression the antigen [peptide or polypeptide] is presentedon the surface of the virus, phage or bacterial host. Libraries of suchparticles that express diverse or families of proteins on their surfaceshave been prepared. The resulting library is then screened with atargeted antigen [receptor or ligand] and those viruses with the highestaffinity for the targeted antigen [receptor or ligand] are selected[see, e.g., U.S. Pat. Nos. 5,403,484, 5,395,750, 5,382,513, 5,316,922,5,288,622, 5,223,409, 5,223,408 and 5,348,867].

Libraries of antibodies expressed on the surfaces of such packages havebeen prepared from spleens of immunized and unimmunized animals and fromhumans. In the embodiment in which a library of phage displayingantibodies from unimmunized human spleens is prepared, it is often ofinterest to screen this library against a large number of differentantigens to identify a number of useful human antibodies for medicalapplications. Phage displaying antibody binding sites derived fromsingle or small numbers of spleen cells can be separately produced,expanded into large batches, and bound to matrices with memories, suchas programmable PROM or EEPROM memories, and identified according tophage batch number recorded in the memory. Each antigen can then beexposed to a large number of different phage-containing memory devices,and those that bind the antigen can be identified by one of severalmeans, including radiolabeled, fluorescent labeled, enzyme labeled oralternate (e.g., mouse) tagged antibody labeled antigen. The encodedinformation in the thus identified phage-containing devices, relates tothe batch of phage reactive with the antigen.

Libraries can also be prepared that contain modified binding sites orsynthetic antibodies. DNA molecules, each encoding proteins containing afamily of similar potential binding domains and a structural signalcalling for the display of the protein on the outer surface of aselected viral or bacterial or other package, such as a bacterial cell,bacterial spore, phage, or virus are introduced into the bacterial host,virus or phage. The protein is expressed and the potential bindingdomain is displayed on the outer surface of the particle. The cells orviruses bearing the binding domains to which target molecules bind areisolated and amplified, and then are characterized. In one embodiment,one or more of these successful binding domains is used as a model forthe design of a new family of potential binding domains, and the processis repeated until a novel binding domain having a desired affinity forthe target molecule is obtained. For example, libraries of de novosynthesized synthetic antibody library containing antibody fragmentsexpressed on the surface have been prepared. DNA encoding syntheticantibodies, which have the structure of antibodies, specifically Fab orFv fragments, and contain randomized binding sequences that maycorrespond in length to hypervariable regions [CDRs] can be insertedinto such vectors and screened with an antigen of choice.

Synthetic binding site libraries can be manipulated and modified for usein combinatorial type approaches in which the heavy and light chainvariable regions are shuffled and exchanged between synthetic antibodiesin order to affect specificities and affinities. This enables theproduction of antibodies that bind to a selected antigen with a selectedaffinity. The approach of constructing synthetic single chain antibodiesis directly applicable to constructing synthetic Fab fragments which canalso be easily displayed and screened. The diversity of the syntheticantibody libraries can be increased by altering the chain lengths of theCDRs and also by incorporating changes in the framework regions that mayaffect antibody affinity. In addition, alternative libraries can begenerated with varying degrees of randomness or diversity by limitingthe amount of degeneracy at certain positions within the CDRs. Thesynthetic binding site can be modified further by varying the chainlengths of the CDRs and adjusting amino acids at defined positions inthe CDRs or the framework region which may affect affinities. Antibodiesidentified from the synthetic antibody library can easily be manipulatedto adjust their affinity and or effector functions. In addition, thesynthetic antibody library is amenable to use in other combinatorialtype approaches. Also, nucleic acid amplification techniques have madeit possible to engineer humanized antibodies and to clone theimmunoglobulin [antibody] repertoire of an immunized mouse from spleencells into phage expression vectors and identify expressed antibodyfragments specific to the antigen used for immunization [see, e.g., U.S.Pat. No. 5,395,750].

The phage or other particles, containing libraries of modified bindingsites, can be prepared in batches and linked to matrices that identifythe DNA that has been inserted into the phage. The matrices are thenmixed and screened with labeled antigen [e.g., fluorescent or enzymatic]or hapten, using an assay carried out with limiting quantities of theantigen, thereby selecting for higher affinity phage. Thus, libraries ofphage linked to matrix particles with memories can be prepared. Thematrices are encoded to identify the batch number of the phage, asublibrary, or to identify a unique sequence of nucleotides or aminoacids in the antibody or antibody fragment expressed on its surface. Thelibrary is then screened with labeled antigens. The antigens are labeledwith enzyme labels or radiolabels or with the antigen bound with asecond binding reagent, such as a second antibody specific for a secondepitope to which a fluorescent antigen binds.

Following identification of antigen bound phage, the matrix particle canbe queried and the identity of the phage or expressed surface protein orpeptide determined. The resulting information represents a profile ofthe sequence that binds to the antigen. This information can be analyzedusing methods known to those of skill in this art.

e. Anti-Microbial Assays and Mutagenicity Assays

Compounds are synthesized or linked to matrix with memory. The linkageis preferably a photocleavable linkage or other readily cleavablelinkage. The matrices with memories with linked compounds, whoseidentities are programmed into each memory are the placed on, forexample, 10 cm culture plates, containing different bacteria, fungi, orother microorganism. After release of the test compounds theanti-microbial effects of the chemical will be assessed by looking forlysis or other indicia of anti-microbial activity. In preferredembodiments, arrays of memories with matrices can be introduced intoplates. The memories are encoded with the identity of the linked orassociated test compound and the position on the array.

The AMES test is the most widely used mutagen/carcinogen screening assay[see, e.g., Ames et al. (1975) Mutation Res. 31:347-364; Ames et al.(1973) Proc. Natl. Acad. Sci. U.S.A. 70:782-786.; Maron et al., (1983)Mutation Research 113:173; Ames (1971) in Chemical Mutagens, Principlesand Methods for their Detection, Vol. 1, Plenum Press, NY, pp 267-282].This test uses several unique strains of Salmonella typhimurium that arehistidine-dependent for growth and that lack the usual DNA repairenzymes. The frequency of normal mutations that render the bacteriaindependent of histidine [i.e., the frequency of spontaneous revertants]is low. The test evaluates the impact of a compound on this revertantfrequency. Because some substances are converted to a mutagen bymetabolic action, the compound to be tested is mixed with the bacteriaon agar plates along with the liver extract. The liver extract serves tomimic metabolic action in an animal. Control plates have only thebacteria and the extract. The mixtures are allowed to incubate. Growthof bacteria is checked by counting colonies. A test is positive wherethe number of colonies on the plates with mixtures containing a testcompound significantly exceeds the number on the corresponding controlplates.

A second type of Ames test [see, International PCT Application No. WO95/10629, which is based on U.S. application Ser. No. 08/011,617; andGee et al. (1994) Proc. Natl. Acad. Sci. U.S.A. 91:11606-11610;commercially avail from Xenometrix, Boulder Colo.] is of interestherein. This test provides a panel of Salmonella typhimurium strains foruse as a detection system for mutagens that also identifies mutagenicchanges. Although a direct descendant of the traditional Ames Salmonellareverse mutation assay in concept, the Ames II assay provides the meansto rapidly screen for base mutations through the use of a mixture of sixdifferent Salmonella strains.

These new strains carry his mutations listed in the table below. All aredeleted for uvrB and are deficient therefore in excision repair. Inaddition, all six have lipopolysaccharide [rfa] mutations rendering themmore permeable, and all contain the pKM¹⁰¹ plasmid conferring enhancedmutability. STRAIN BASE CHANGE MUTATION TA7001 A:T → G:C hisG1775 TA7002T:A → A:T hisC9138 TA7003 T:A → G:C hisG9074 TA7004 G:C → A:T hisG9133TA7005 G:C → A:T hisG9130 TA7006 G:C → C:G hisC9070

These strains, which revert at similar spontaneous frequencies[approximately 1 to 10×10⁸] can be exposed and plated separately fordetermining mutational spectra, or mixed and exposed together to assessbroad mutagenic potential. The assay takes 3 days from start to finishand can be performed in 96 well- or 384 well-microtiter plates.Revertant colonies are scored using bromo-creosol purple indicator dyein the growth medium. The mixed strains can be assayed first as part ofa rapid screening program. Since this six strain mixture is slightlyless sensitive than individual strains tested alone, compounds which arenegative for the mix can be retested using all six strains. For all butthe weakest mutagens, the Ames II strain mixture appears to be capableof detecting reversion events even if only one strain is induced torevert. The mixed strains provide a means to perform rapid initialscreening for genotoxins, while the battery of base-specific testerstrains permit mutational spectra analysis.

As modified herein, the test compounds are linked to matrices withmemories, that have been encoded with the identity of the testcompounds. The assays can be performed on multiple test compoundssimultaneously using arrays of matrices with memories or multiplematrices with memories encoded with the identity of the linked testcompound and the array position or plate number into which the compoundis introduced.

f. Hybridization Assays and Reactions

(1) Hybridization Reactions

It is often desirable to detect or quantify very small concentrations ofnucleic acids in biological samples. Typically, to perform suchmeasurements, the nucleic acid in the sample [i.e., the target nucleicacid] is hybridized to a detection oligonucleotide. In order to obtain adetectable signal proportional to the concentration of the targetnucleic acid, either the target nucleic acid in the sample or thedetection oligonucleotide is associated with a signal generatingreporter element, such as a radioactive atom, a chromogenic orfluorogenic molecule, or an enzyme [such as alkaline phosphatase] thatcatalyzes a reaction that produces a detectable product. Numerousmethods are available for detecting and quantifying the signal.

Following hybridization of a detection oligonucleotide with a target,the resulting signal-generating hybrid molecules must be separated fromunreacted target and detection oligonucleotides. In order to do so, manyof the commonly used assays immobilize the target nucleic acids ordetection oligonucleotides on solid supports. Presently available solidsupports to which oligonucleotides are linked include nitrocellulose ornylon membranes, activated agarose supports, diazotized cellulosesupports and non-porous polystyrene latex solid microspheres. Linkage toa solid support permits fractionation and subsequent identification ofthe hybridized nucleic acids, since the target nucleic acid may bedirectly captured by oligonucleotides immobilized on solid supports.More frequently, so-called “sandwich” hybridization systems are used.These systems employ a capture oligonucleotide covalently or otherwiseattached to a solid support for capturing detectionoligonucleotide-target nucleic acid adducts formed in solution [see,e.g., EP 276,302 and Gingeras et al. (1989) Proc. Natl. Acad. Sci. USA86:1173]. Solid supports with linked oligonucleotides are also used inmethods of affinity purification. Following hybridization or affinitypurification, however, if identification of the linked molecule orbiological material is required, the resulting complexes or hybrids orcompounds must be subjected to analyses, such as sequencing. Thecombinations and methods herein eliminate the need for such analyses.

Use of matrices with memories in place of the solid support matricesused in the prior hybridization methods permits rapid identification ofhybridizing molecules. The identity of the linked oligonucleotide iswritten or encoded into the memory. After reaction, hybrids areidentified, such as by radioactivity or separation, and the identify ofhybridizing molecules are determined by querying the memories.

(2) Hybridization Assays

Mixtures nucleic acid probes linked to the matrices with memories can beused for screening in assays that heretofore had to be done with oneprobe at a time or with mixtures of probes followed by sequencing thehybridizing probes. There are numerous examples of such assays [see,e.g., U.S. Pat. No. 5,292,874, “Nucleic acid probes to Staphylococcusaureus” to Milliman, and U.S. Pat. No. 5,232,831, “Nucleic acid probesto Streptococcus pyogenes” to Milliman, et al.; see, also, U.S. Pat.Nos. 5,216,143, 5,284,747 5,352,579 and 5,374,718]. For example, U.S.Pat. No. 5,232,831 provides probes for the detection of particularStreptococcus species from among related species and methods using theprobes. These probes are based on regions of Streptococcus rRNA that arenot conserved among related Streptococcus species. Particular speciesare identified by hybridizing with mixtures of probes and ascertainingwhich probe(s) hybridize. By virtue of the instant matrices withmemories, following hybridization, the identity of the hybridizingprobes can be determined by querying the memories, and therebyidentifying the hybridizing probe.

i. Combinatorial Libraries and Other Libraries and ScreeningMethodologies

The combinations of matrices with memories are applicable to virtuallyany synthetic scheme and library preparation and screening protocol.These include, those discussed herein, and also methodologies anddevices, such as the Chiron “pin” technology [see, e.g., InternationalPCT application No. WO 94/11388; Geysen et al. (1985) Proc. Natl. Acad.Sci. U.S.A. 82:178; and Geysen et al. (1987) J. Immunol. Meth.102:259-274] which relies on a support composed of annular synthesiscomponents that have an active surface for synthesis of a modularpolymer and an inert support rod that is positioned axially to theannular synthesis components. This pin technology was developed for thesimultaneous synthesis of multiple peptides. In particular the peptidesare synthesized on polyacrylic acid grafted on the tip of polyethylenepins, typically arranged in a microtiter format. Amino acid coupling iseffected by immersing the pins in a microtiter plate. The resultingpeptides remain bound to the pins and can be reused.

As provided herein, “pins” may be linked to a memory or recordingdevice, preferably encasing the device, or each pin may be coded and thecode and the identity of the associated linked molecule(s) stored in aremote memory. As a result it will not be necessary to physically arraythe pins, rather the pins can be removed and mixed or sorted.

Also of interest herein, are DIVERSOMER™ technology libraries producedby simultaneous parallel synthesis schemes for production ofnonoligomeric chemical diversity [see, e.g., U.S. Pat. No. 5,424,483;Hobbs DeWitt et al. (1994) Drug Devel. Res. 33:116-124; Czarnik et al.(1994) Polym. Prepr. 35:985; Stankovic et al. (1994) in InnovationPerspect. Solid Phase Synth. Collect. Pap. Int. Symp., 3rd Epton, R.(Ed), pp. 391-6; DeWitt et al. (1994) Drug Dev. Res. 33:116-124; HobbsDeWitt et al. (1993) Proc. Natl. Acad. Sci. U.S.A. 90:6909-6913]. Inthis technology a starting material is bonded to a solid phase, such asa matrix material, and is subsequently treated with reagents in astepwise fashion. Because the products are linked to the solid support,multistep syntheses can be automated and multiple reactions can beperformed simultaneously to produce libraries of small molecules. Thistechnology can be readily improved by combining the matrices withmemories or encoding the matrix supports in accord with the methodsherein.

The matrices with memories, either those with memories in proximity orthose in which the matrix includes a code stored in a remote memory, canbe used in virtually any combinatorial library protocol. These protocolsor methodologies and libraries, include but are not limited to thosedescribed in any of following references: Zuckermann et al. (1994) J.Med. Chem. 37:2678; Martin et al. (1995) J. Med. Chem. 38:1431; Campbellet al (1995) J. Am. Chem. Soc. 117:5381; Salmon et al. (1993) Proc.Natl. Acad. Sci. U.S.A. 90:11708: Patek et al. (1994) Tetrahedron Lett.35:9169; Patek et al. (1995) Tetrahedron Lett. 36:2227; Hobbs DeWitt etal. (1993) Proc. Natl. Acad. Sci. U.S.A. 90:6906; Baldwin et al. (1995)J. Am. Chem. Soc. 117:5588; and any others.

h. Nucleic Acid Sequencing

Methods of DNA sequencing based on hybridization of DNA fragments with acomplete set of fixed length oligonucleotides [8-mers] that areimmobilized individually as dots in a 2-dimensional matrix is sufficientfor computer-assisted reconstruction of the sequences of fragments up to200 bases long [International PCT Application WO 92/10588]. The nucleicacid probes are of a length shorter than a target, which is hybridizedto the probes under conditions such that only those probes having anexact complementary sequence are hybridized maximally, but those withmismatches in specific locations hybridize with a reduced affinity, ascan be determined by conditions necessary to dissociate the pairs ofhybrids. Alignment of overlapping sequences from the hybridizing probesreconstructs the complement of the target [see, EP 0 535 242 A1,International PCT Application WO 95/00530, and Khrapko et al. (1989)FEBS Lttrs. 256:118-122]. The target fragment with the sequence ofinterest is hybridized, generally under highly stringent conditions thattolerate no mismatches or as described below a selected number ofmismatches, with mixtures of oligonucleotides [typically a mixture ofoctomers of all possible sequences] that are each immobilized on amatrix with memory that is encoded with the sequence of the probe. Uponhybridization, hybridizing probes are identified by routine methods,such as OD or using labeled probe, and the sequences of the hybridizingprobes can be determined by retrieving the sequences from the linkedmemories. When hybridization is carried out under conditions in which nomismatches are tolerated, the sequence of the target can then bedetermined by aligning overlapping sequences of the hybridizing probes.

Previous methods used to accomplish this process have incorporatedmicroscopic arrays of nucleotide oligomers synthesized on small siliconbased chips. It is difficult to synthesize such arrays and qualitycontrol the large number of spots on each chip (about 64,000 spots for8-mer oligonucleotides, that number necessary to accomplish sequencingby hybridization). In the present method, each oligomer is independentlysynthesized on a batch of individual chips, those chips are tested foraccuracy and purity of their respective oligomers, then one chip fromeach batch is added to a large pool containing oligomers having allpossible sequences. After hybridization in batch mode with the genesegment to be sequenced, usually amplified by a method such as PCR,using appropriate primers, and labeled with a detectable [such asfluorescent] tag, the chips can be passed through a detector, such asdescribed above for processing multiplexed assays, including multiplexedimmunoassays, and the degree of binding to each oligomer can bedetermined. After exposing the batch to varying degrees of dissociatingconditions, the devices can again be assayed for degree of binding, andthe strength of binding to related sequences will relate the sequence ofthe gene segment [see, e.g., International PCT Application WO 95/00530].

j. Separations, Physical Mapping and Measurements of Kinetics of Bindingand Binding Affinities

Multiple blots [i.e., Western, Northern, Southern and/or dot blots] maybe simultaneously reacted and processed. Each memory, in the form of arectangle or other suitable, is linked or coated on one surface withmaterial, such as nitrocellulose, to which or the analyte of interestbinds or with which it reacts. The chips are arranged in an array, suchas in strips that can be formed into rectangles or suitable othershapes, circles, or in other geometries, and the respective x-ycoordinate or other position-identifying coordinate(s), and, if needed,sheet number and/or other identifying information, is programmed intoeach memory. Alternatively, they may be programmed with thisidentification, then positioned robotically or manually into an arrayconfiguration. They are preferably linked together, such as byreversible glue, or placing them in agarose, or by any suitable methodas long as the reactive surface is not disturbed. Following transfer ofthe material, such as transfer of protein from a Western Blot, nucleicacid from a Southern or Northern blot, dot blots, replica platedbacterial culture, or viral plaques, the memories are separated andmixed for reaction with a traditionally labeled, such as a fluorescentlabel, detection nucleic acid, protein, antibody or receptor ofinterest. Complexes are identified, and their origin in the blotdetermined by retrieving the stored information in each chip.Quantitation may also be effected based on the amount of label bound.

A series of appropriately activated matrices with memories are arrangedin an array, one or, preferably two dimensional. In one configuration,each chip is pre-programmed and placed in a specific location that isentered into its memory, such as an x-y coordinate. At least one surfaceof the memory with matrix is treated so that the transferred reagentbinds. For example, a piece of nitrocellulose can be fixed to one sideof the memory device. The resulting array is then contacted with aseparation medium whereby each reagent of interest is transferred to andbound to the end of the matrix with memory such that the reagentlocation is known. The matrices are separated and pooled; multiplearrays may be pooled as long as source information is recorded in eachmemory. All matrices with memories are then contacted with detectionagents that specifically bind to reagents in the mixture. The matriceswith memories are passed through a reading device, either after anincubation for end point determinations or continuously for kineticmeasurements. The reading devices is a device that can detect label,such as fluorescence, and an reader, such as an RF ready, that can querythe memory and identify each matrix. The rate of binding and maximumbinding and identify of bound reagents can be determined.

Dot blots, for example, can be used in hybridoma analysis to identifyclones that secrete antibodies of desired reactivity and to determinethe relative affinities of antibodies secreted by different cell lines.Matrices with memories that are activated to bind immunoglobulins andwith on-board information specifying their relative locations in thearray are dipped in an array into the wells of microplates containinghybridoma cells. After incubation, they are withdrawn, rinsed, removedand exposed to labeled antigen. Matrices of desired specificity andaffinity are selected and read thereby identifying the original wellscontaining the hybridoma cells that produce the selected antibodies.

In other embodiments, the transfer medium [i.e., the nitrocellulose orother such medium] may be part of the surface of the chip or array ofchips that can bind to the separated species subsequent to separation.For example, the separation system, such as the agarose orpolyacrylamide gel, can be included on the surface(s) of the matrix withmemories in the array. After separation the surface will be activatedwith a photoactivatable linker or suitable activating agent to therebycovalently link, such as by a photoflash, the separated molecules to thematrices in the array.

Alternatively, each matrix with memory may have one or more specificbinding agents, such as an antibody or nucleic acid probe, attached(adsorbed, absorbed, or otherwise in physical contact) to matrix withmemory. The matrix with memory and linked binding agent is thencontacted with a medium containing the target(s). After contacting,which permits binding of any targets to which the linked binding agentsspecifically bind, the matrix with memory is processed to identifymemories with matrices to which target has specifically bound viainteraction with the binding agent. For example, the (1) the target islabeled, thereby permitted direct detection of complexes; (2) the memorywith matrix is then contacted with a developing agent, such as a secondantibody or detection probe, whereby binding agent-target complexes aredetected; or (3) the detection agent is present during the reaction,such as non-specifically attached to the matrix with memory or by othermethod [thin film, coated on the matrix with memory, coated onnitrocellulose].

Such support bound analytes may also be used to analyze the kinetics ofbinding by continuously passing the supports through a label readingdevice during the reaction, and identify the labeled complexes. Thebinding agents can be eluted, either in a kinetically readable manner orin batch. In addition, since the recording devices may also includecomponents that record reaction conditions, such as temperature and pH,kinetics, which are temperature and pH dependent, may be accuratelycalculated.

After elution, the support bound analytes may be identified to analyzekinetics of binding to the binding agent. Such binding and elutionprotocols may also be adapted to affinity purification methodologies.

k. Cell Sorting

The devices herein may also be used in methods of cell sorting. Forexample, the memory with matrix combinations are linked to selectedantigens, information regarding the antigens is encoded into thememories, the resulting combinations are used in multi-analyte analysesof cells.

It is possible to identify a profile of cells exhibiting differentsurface markers [antigens, for example, or other ligands or receptormolecules] by using combinations of labeled and matrix memory-boundbinding agents. In one embodiment, each agent, such as an antibody,capable of binding specifically to one of many different surface markersis bound to a different matrix with a memory. The nature of therecognized marker is recorded in the memory of each matrix-binding agentcomplex, and the mixture of binding-agent-matrix memory complexes isreacted with a mixture of cells. The cell-matrix complexes that resultfrom binding agents attaching cells to the surfaces of the respectivematrices are then reacted with a labeled [for example, fluorescent]reagent or mixture of reagents which also reacts with the cells. Theselabeled reagents can be the same or different from those coupled to thememory matrices. When the matrices are passed through a reader [to readthe label and the memory], those that have bound cells can be identifiedand if necessary isolated. This application is particularly useful forscreening for rare cells, for example stem cells in a bone marrow orperipheral lymphocyte sample, for detecting tumor cells in a bone marrowsample to be used for autologous transplantation, or for fetal cells ina maternal circulation.

In these embodiments, the memory with matrices herein can be counted andread with instruments, such as a device that operates on the principlesof a Coulter counter, that are designed to count cells or particles. Inusing a Coulter Counter, a suspension of cells or particles is suckedthrough a minute hole in a glass tube. One electrode is placed withinthe tube and another is outside of the tube in the suspension. Thepassage of a particle through the hole temporarily interrupts thecurrent; the number of interruptions is determined by a conventionalscaling unit.

For use herein, such instruments are modified by including an RF reader[or other reader if another frequency or memory means is selected] sothat the identity of the particle or cell [or antigen on the cell orother encoded information] can be determined as the particle or cellpasses through the hole and interrupts the current, and also, if needed,a means to detect label, such as fluorescent label. As the particlepasses through the hole the RF reader will read the memory in the matrixthat is linked to the particle. The particles also may be countedconcurrently with the determination of the identity of the particle.Among the applications of this device and method, is a means to sortmultiple types of cells at once.

l. Drug Delivery and Detecting Changes in Internal Conditions in theBody

Memories may also be combined with biocompatible supports and polymersthat are used internally in the bodies of animals, such as drug deliverydevices [see, e.g., U.S. Pat. Nos. 5,447,533, 5,443,953, 5,383,873,5,366,733, 5,324,324, 5,236,355, 5,114,719, 4,786,277, 4,779,806,4,705,503, 4,702,732, 4,657,543, 4,542,025, 4,530,840, 4,450,150 and4,351,337] or other biocompatible support [see, U.S. Pat. No. 5,217,743and U.S. Pat. No. 4,973,493, which provide methods for enhancing thebiocompatibility of matrix polymers]. Such biocompatible polymersinclude matrices of poly(ethylene-co-vinyl acetate) and matrices of apolyanhydride copolymer of a stearic acid dimer and sebacic acid [see,e.g., Sherwood et al. (1992) Bio/Technology 10:1446-1449].

The biocompatible drug delivery device in combination with the memory isintroduced into the body. The device, generally by virtue of combinationwith a biosensor or other sensor, also monitors pH, temperature,electrolyte concentrations and other such physiological parameters andin response to preprogrammed changes, directs the drug delivery deviceto release or not release drugs or can be queried, whereby the change isdetected and drug delivered or administered.

Alternatively, the device provided in combination with a biocompatiblesupport and biosensor, such that the information determined by thebiosensor can be stored in the device memory. The combination of deviceand biosensor is introduced into the body and is used to monitorinternal conditions, such as glucose level, which level is written tomemory. The internal condition, such as glucose level, electrolytes,particularly potassium, pH, hormone levels, and other such level, canthen be determined by querying the device.

In one embodiment, the device, preferably one containing a volatilememory that is read to and written using RF, linked to a biosensor [see,e.g., U.S. Pat. No. 5,384,028 which provides a biosensor with a datamemory that stores data] that can detect a change in an internalcondition, such as glucose or electrolyte, and store or report thatchange via RF to the linked matrix with memory, which records suchchange as a data point in the memory, which can then be queried. Theanimal is then scanned with RF and the presence of the data point isindicative of a change. Thus, instead of sampling the body fluid, thememory with matrix with linked biosensor is introduced into a site inthe body, and can be queried externally. For example, the sensor can beembedded under the skin and scanned periodically, or the scanner is wornon the body, such as on the wrist, and the matrix with memory eitherperiodically, intermittently, or continuously sends signals; the scanneris linked to an infusion device and automatically, when triggeredtriggers infusion or alters infusion rate.

m. Multiplexed or Coupled Protocols in which the Synthesis Steps [theChemistry] is Coupled to Subsequent Uses of the Synthesized Molecules

Multiplexed or multiple step processes in which compounds aresynthesized and then assayed without any intermediate identificationsteps are provided herein. Since the memories with matrices permitidentification of linked or proximate or associated molecules orbiological particles, there is no need to identify such molecules orbiological particles during any preparative and subsequent assayingsteps or processing steps. Thus, the chemistry [synthesis] can bedirectly coupled to the biology [assaying, screening or any otherapplication disclosed herein]. For purposes herein this coupling isreferred to as multiplexing. Thus, high speed synthesis can be coupledto high throughput screening protocols.

F. Applications of the Memories with Matrices and Luminescing Matriceswith Memories in Combinatorial Syntheses and Preparation of Libraries

Libraries of diverse molecules are critical for identification of newpharmaceuticals. A diversity library has three components: solid supportmatrix, linker and synthetic target. The support is a matrix material asdescribed herein that is stable to a wide range of reaction conditionsand solvents; the linker is selectively cleavable and does not leave afunctionalized appendage on the synthetic target; and the target issynthesized in high yield and purity. For use herein, the diversitylibrary further includes a memory or recording device in combinationwith the support matrix. The memory is linked, encased, in proximitywith or otherwise associate with each matrix particle, whereby theidentify of synthesized targets is written into the memory.

The matrices with memories are linked to molecules and particles thatare components of libraries to electronically tagged combinatoriallibraries. Particularly preferred libraries are the combinatoriallibraries that containing matrices with memories that employ radiofrequencies for reading and writing.

1. Oligomer and Polypeptide Libraries

a. Bio-Oligomer Libraries

One exemplary method for generating a library [see, U.S. Pat. No.5,382,513] involves repeating the steps of (1) providing at least twoaliquots of a solid phase support: separately introducing a set ofsubunits to the aliquots of the solid phase support; completely couplingthe subunit to substantially all sites of the solid phase support toform a solid phase support/new subunit combination, assessing thecompleteness of coupling and if necessary, forcing the reaction tocompleteness; thoroughly mixing the aliquots of solid phase support/newsubunit combination; and, after repeating the foregoing steps thedesired number of times, removing protecting groups such that thebio-oligomer remains linked to the solid phase support. In oneembodiment, the subunit may be an amino acid, and the bio-oligomer maybe a peptide. In another embodiment, the subunit may be a nucleoside andthe bio-oligomer may be an oligonucleotide. In a further embodiment, thenucleoside is deoxyribonucleic acid; in yet another embodiment, thenucleoside is ribonucleic acid. In a further embodiment, the subunit maybe an amino acid, oligosaccharide, oligoglycosides or a nucleoside, andthe bio-oligomer may be a peptide-oligonucleotide chimera or otherchimera. Each solid phase support is attached to a single bio-oligomerspecies and all possible combinations of monomer [or multimers incertain embodiments] subunits of which the bio-oligomers are composedare included in the collection.

In practicing this method herein, the support matrix has a recordingdevice with programmable memory, encased, linked or otherwise attachedto the matrix material, and at each step in the synthesis the supportmatrix to which the nascent polymer is attached is programmed to recordthe identity of the subunit that is added. At the completion ofsynthesis of each biopolymer, the resulting biopolymers linked to thesupports are mixed.

After mixing an acceptor molecule or substrate molecule of interest isadded. The acceptor molecule is one that recognizes and binds to one ormore solid phase matrices with memory/bio-oligomer species within themixture or the substrate molecule will undergo a chemical reactioncatalyzed by one or more solid phase matrix with memory/bio-oligomerspecies within the library. The resulting combinations that bind to theacceptor molecule or catalyze reaction are selected. The memory in thematrix-memory combination is read and the identity of the activebio-oligomer species is determined.

b. Split Bead Sequential Syntheses

Various schemes for split bead syntheses of polymers [FIG. 1], peptides[FIG. 2], nucleic acids [FIG. 3] and organic molecules based on apharmacophore monomer [FIG. 4] are provided. Selected matrices withmemory particles are placed in a suitable separation system, such as afunnel [see, FIG. 5]. After each synthetic step, each particle isscanned [i.e., read] as it passes the RF transmitter, and informationidentifying the added component or class of components is stored inmemory. For each type of synthesis a code can be programmed [i.e., a 1at position 1,1 in the memory could, for example, represent alanine atthe first position in the peptide]. A host computer or decoder/encoderis programmed to send the appropriate signal to a transmitter thatresults in the appropriate information stored in the memory [i.e., foralanine as amino acid 1, a 1 stored at position 1,1]. When read, thehost computer or decoder/encoder can interpret the signal read from andtransmitted from the memory.

In an exemplary embodiment, a selected number of beads [, particulatematrices with memories [matrix particles linked to recording devices],typically at least 10³, more often 10⁴, and desirably at least 10⁵ ormore up to and perhaps exceeding 10¹⁵, are selected or prepared. Thebeads are then divided into groups, depending upon the number of choicesfor the first component of the molecule. They are divided into a numberof containers equal to or less than [for pooled screening, nestedlibraries or the other such methods] the number of choices. Thecontainers can be microtiter wells, Merrifield synthesis vessels,columns, test tubes, gels, etc. The appropriate reagents and monomer areadded to each container and the beads in the first container are scannedwith electromagnetic with radiation, preferably high frequency radiowaves, to transmit information and encode the memory to identify thefirst monomer. The beads in the second container are so treated. Thebeads are then combined and separated according to the combinatorialprotocol, and at each stage of added monomer each separate group islabeled by inputting data specific to the monomer. At the end of thesynthesis protocol each bead has an oligomer attached and informationidentifying the oligomer stored in memory in a form that can beretrieved and decoded to reveal the identity of each oligomer.

An 8-member decapeptide library was designed, synthesized, and screenedagainst an antibody specifically generated against one of the librarymembers using the matrices with memories. Rapid and clean encoding anddecoding of structural information using radio frequency signals,coupling of combinatorial chemical synthesis to biological assayprotocols, and potential to sense and measure biodata using suitablebiosensors, such as a temperature thermistor or pH electrode, embeddedwithin the devices have been demonstrated. The “split and pool” method[see, e.g., Furka et al. (19910 Int. J. Pept. Protein Res. 37:487-493;Lam et al. (1991) Nature 354:82-84; and Sebestyén et al. (1993) Bioorg.Med. Chem. Lett. 3:413-418] was used to generate the library. An ELISA[see e.g., Harlow et al. (1988) Antibodies, a laboratory manual, ColdSpring Harbor, N.Y.] was used to screen the library for the peptidespecific for the antibody.

2. “Nested” Combinatorial Library Protocols

In this type of protocol libraries of sublibraries are screened, and asublibrary selected for further screening [see, e.g., Zuckermann et al.(1994) J. Med. Chem. 37:2678-2685; and Zuckermann et al. (1992) J. Am.Chem. Soc. 114:10646-10647]. In this method, three sets of monomers werechosen from commercially available monomers, a set of four aromatichydrophobic monomers, a set of three hydroxylic monomers, a set ofseventeen diverse monomers, and three N-termini were selected. Theselection was based on an analysis of the target receptor and knownligands. A library containing eighteen mixtures, generated from the sixpermutations of the three monomer sets, times three N-termini wasprepared. Each mixture of all combinations of the three sets of amines,four sets of hydrophobic monomers and seventeen diverse monomers wasthen assayed. The most potent mixture was selected for deconvolution bysynthesis of pools of combinatorial mixtures of the components of theselected pool. This process was repeated, until individual compoundswere selected.

Tagging the mixtures with the matrices with memories will greatlysimplify the above protocol. Instead of screening each mixtureseparately, each matrix particle with memory will be prepared with setsof the compounds, analogous to the mixtures of compounds. The resultingmatrix particles with memories and linked compounds can be combined andthen assayed. As with any of the methods provided herein, the linkedcompounds [molecules or biological particles] can be cleaved from thematrix with memory prior to assaying or anytime thereafter, as long asthe cleaved molecules remain in proximity to the device or in somemanner can be identified as the molecules or particles that were linkedto the device. The matrix particle(s) with memories that exhibit thehighest affinity [bind the greatest amount of sample at equilibrium] areselected and identified by querying the memory to identify the group ofcompounds. This group of compounds is then deconvoluted and furtherscreened by repeating this process, on or off the matrices withmemories, until high affinity compounds are selected.

3. Other Combinatorial Protocols

The matrices with memories provided herein may be used as supports inany synthetic scheme and for any protocol, including protocols forsynthesis of solid state materials. Combinatorial approaches have beendeveloped for parallel synthesis of libraries of solid state materials[see, e.g. Xiang et al. (1995) Science 268:1738-1740]. In particular,arrays containing different combinations, stoichiometries, anddeposition sequences of inorganics, such as BaCO₃, BiO₃, CaO, CuO, PbO,SrCO₃ and Y₂O₃, for screening as superconductors have been prepared.These arrays may be combined with memories that identify position andthe array and/or deposited material.

The following examples are included for illustrative purposes only andare not intended to limit the scope of the invention.

EXAMPLE 1

Formulation of a Polystyrene Polymer on Glass and Derivatization ofPolystyrene

A glass surface of any conformation [beads for exemplification purposes(1)] that contain a selected memory device that coat the device or thatcan be used in proximity to the device or subsequently linked to thedevice is coated with a layer of polystyrene that is derivatized so thatit contains a cleavable linker, such as an acid cleavable linker. Toeffect such coating a bead, for example, is coated with a layer of asolution of styrene, chloromethylated styrene, divinyl benzene, benzoylperoxide [88/10/1/1/, molar ratio] and heated at 70° C. for 24 h. Theresult is a cross-linked chloromethylated polystyrene on glass (2).Treatment of (2) with ammonia [2 M in 1,4-dioxane, overnight] producesaminomethylated coated beads (3). The amino group on (3) is coupled withpolyethylene glycol dicarboxymethyl ether (4) [n≈20] under standardconditions [PyBop/DIEA] to yield carboxylic acid derivatized beads (5).Coupling of (5) with modified PAL [PAL is pyridylalanine] linker (6)under the same conditions produces a bead that is coated withpolystyrene that has an acid cleavable linker (7).

The resulting coated beads with memories are then used as solid supportfor molecular syntheses or for linkage of any desired substrate.

EXAMPLE 2

Construction of a Matrix with Memory

A matrix with memory was constructed from (a) and (b) as follows:

(a) A small (8×1×1 mm) semiconductor memory device [the IPTT-100purchased from Bio Medic Data Systems, Inc., Maywood, N.J.; see, alsoU.S. Pat. Nos. 5,422,636, 5,420,579, 5,262,772, 5,252,962, 5,250,962,5,074,318, and RE 34,936].

The memory device is a transponder [IPTT-100, Bio Medic Data Systems,Inc., Maywood, N.J.] that includes a remotely addressable memory[EEPROM]. The transponder receives, stores and emits radio frequencysignals of different frequencies so that it can be remotely programmedwith information regarding synthetic steps and the constituents oflinked or proximate molecules or biological particles.

These devices are designed to operate without a battery, relying on theenergy generated by the radio frequency pulses used in the encodingprocess. Also, it is important to note that additional sensors such astemperature [as in this case], pH, or concentration measuring devicescan be installed. The resulting combinations are capable of withstandingmost reagents and conditions used in synthetic organic chemistry,including temperatures from −78 to 150° C.

The transponder was encoded and read with a device that emits and readsRF frequencies [Bio Medic Data Systems Inc. DAS-5001 CONSOLE™ System,see, also U.S. Pat. No. 5,252,962].

These memory devices include EEPROM (Electrical, Erasable, Programmable,Read-Only Memory) “flash” unit and a temperature sensing device able toaccept or emit information at any time. At each step of thecombinatorial “split and pool” sequence, encoding information is sentfrom a distance in the form of radio frequency pulses at 145 kHz andstored until decoding is needed. When needed the radio frequency signalsare retrieved using a specially assembled apparatus capable of readingthe radio frequency code from a distance [DAS-5001 CONSOLE™ from BioMedic Data Systems, Inc., Maywood, N.J.; see, e.g., U.S. Pat. Nos.5,422,636, 5,420,579, 5,262,772, 5,252,962 and 5,250,962, 5,252,962 and5,262,772].

(b) TENTAGEL™ polymer beads carrying an acid-cleavable linker [TENTAGELS Am cat #S30 022. RAPP Polymer, Tubingen, Germany].

(c) A chemically inert surrounding porous support [polypropylene AA,SPECTRUM, Houston, Tex.].

One transponder and about 20 mg of the derivatized TENTAGEL™ beads havebeen sealed in a small [of a size just sufficient to hold the beads andtransponder] porous polypropylene microvessel [see, Examples 3 and 4].

EXAMPLE 3

Microvessels

A. FIGS. 11-13

FIGS. 11-13 illustrate an embodiment of a microvessel 20 providedherein. The microvessel 20 is a generally elongated body with walls 22of porous or semi-permeable non-reactive material which are sealed atboth ends with one or more solid-material cap assemblies 42, 44. Themicrovessel 20 retains particulate matrix materials 40 and one or morerecording devices 34. In the preferred embodiment illustrated in FIGS.11-13, the recording device includes a shell 36 that is impervious tothe processing steps or solutions with which the microvessel may comeinto contact, but which permits transmission of electromagnetic signals,including radiofrequency, magnetic or optical signals, to and from therecording media of the recording device.

The preferred microvessel 20 is generally cylindrically shaped and hastwo solid-material cap assemblies 42, 44. The cap assemblies may beformed of any material that is non-reactive with the solutions withwhich the microvessel will come into contact. Such appropriate materialsinclude, for example, plastic, teflon, poly-tetra-fluoro-ethylene(hereinafter, PTFE) or polypropylene. Each cap assembly 42, 44preferably includes a support base 26, 28, respectively, and an end cap24, 30, respectively. Each support base 26, 28 is permanently attachedto the walls 22 of the vessel by known means such as bonding withappropriate adhesives or heat treatment, either by heat-shrinking thewall material onto the lower portions of the support bases 26,28, or byfusing the wall material with the support base material.

Preferably, at least one of the caps 24,30 is removably attached to itscap base 26, for example by providing complementary threads on thesupport base and the end cap so that the end cap can be screwed into thesupport base, as illustrated in FIG. 12. Other possible means forattaching the end cap to the support base will be apparent to those inthe art, and can include snap rings, spring tabs, and bayonetconnectors, among others. The end cap 24, has one or more slots, bores,or recesses 32 formed in its outer surface to facilitate removal orreplacement, with the user's fingers and/or by use of an appropriatetool. For the example illustrated, a spanner wrench having pegs spacedat the same separation as the recesses 32 can be used by inserting thepegs into the recesses. For a single slot, removal and replacement ofthe end cap could be achieved by using a screwdriver. Protruding tabs,rims, knurled edges or other means to enhance the ability to grasp theend cap can be used for manual assembly/disassembly of the microvessel.The cap assembly 42 at the opposite end of the microvessel can bepermanently sealed using an adhesive or heat treatment to attach thesupport base 28 to the end cap 30, or the cap assembly 42 can be moldedas a single piece, combining the support base 28 and the end cap 30.

Retained within the microvessel 20 are particle matrix materials 40 anda memory device 34. The recording device 34, in the preferred embodimentillustrated, includes a data storage unit(s) 38 and a shell 36 thatprotects the recording device 38 from the processing steps and/orsolutions to which the microvessel are subjected. This shell 36 ispreferably constructed of material that is non-reactive with andimpervious to the solutions with which the microvessel may come intocontact, and which is penetrable by the electromagnetic radiation, orsimilar means, used to read from and write to the memory device. Thepreferred device is presently a modified form of the IPTT-100 [Bio MedicData Systems, Inc. (“BMDS”), Maywood, N.J.; see, also U.S. Pat. Nos.5,422,636, 5,420,579, 5,262,772, 5,252,962 and 5,250,962], whichgenerally contains an electrically programmable memory chip 130 anddecoding and power conversion circuitry 132 mounted on an elongatedceramic circuit board 134 and connected to an LC oscillator, comprisingcapacitor 138 and coil 136 wound around a ferrite core, whichinductively receives and responds to a frequency-modulated magneticsignal generated by a similar LC oscillator in the write device,allowing the device to be remotely encoded and remotely read at adistance on the order of 1 cm or less. The device has been modified fromthe supplier's standard commercially-available form to provide physicaldimensions to facilitate placement in the microvessel 20. Themodification involves application of the simple and well-knownrelationship between inductor core area and length, the permeability ofthe core material, and the number of windings, i.e., L (inductance)=N²μA/l, where N is the number of windings, μ the permeability of the core,A is the core area and l is the core length.

Other remotely programmable and readable tags are commercially availablewhich may be used in the inventive system, such as those manufactured byIdentification Device Technology, UK. These devices have circuitry andoperational parameters similar to the device described above, but it maybe necessary to modify the coil to reduce the access range to less thanor equal to 1 cm. It is generally preferred that the responder, i.e.,the memory device, and the transceiver in the control system be from thesame manufacturer to assure complete compatibility.

The illustrated microvessel, as illustrated in FIGS. 11-13, is of a sizesufficient to contain at least one recording device and one matrixparticle, such as a TENTAGEL™ bead. The device is typically 20 mm inlength [i.e., the largest dimension] or smaller, with a diameter ofapproximately 5 mm or less, although other sizes are also contemplated.These sizes are sufficient to contain form about 1 mg up to about 1 g ofmatrix particle, and thus range from about 1 mm up 100 mm in the largestdimension, typically about 5 mm to about 50 mm, preferably 10 mm to 30mm, and most preferably about 15 to 25 mm. The size, of course can besmaller than those specified or larger. The wall material of themicrovessel is PTFE mesh having a preferably about 50 μM to 100 μM,generally 50 to 70 μM hole size that is commercially available. The sizeof course is selected to be sufficiently small to retain the matrixparticles. The cap apparatus is machined rod PTFE [commerciallyavailable from McMaster Carr, as Part #8546K11].

The matrix material is selected based upon the particular use of themicrovessel; for example, a functionalized resin, such as TENTAGEL™resin, commercially available from Rapp Polymere, Tubingen, Germany, ispreferred for use in peptide synthesis and similar processes. The matrixmaterial may also include fluophores or scintillants as describedherein.

Alternative embodiments of the microvessel will be appreciated andinclude, for example, a pouch, including porous or semi-permeablematerial, which is permanently sealed to itself and contains matrixmaterial and one or more memories.

B. FIGS. 14-16

FIGS. 14-16 illustrate an alternate embodiment of a microvessel providedherein. Like the microvessel described in Example 3, this embodiment ofthe microvessel also retains particulate matrix materials and one ormore recording devices (not illustrated). The microvessel has asingle-piece solid material frame 82, including a top ring 84, twosupport ribs 88, 100 disposed diametrically opposite each other and abottom cap 86. The solid material frame 82 may be constructed of anymaterial which is non-reactive with the solutions with which themicrovessel will come into contact. Such appropriate materials include,for example, plastic, teflon, poly-tetra-fluoro-ethylene (hereinafter,PTFE) or polypropylene, and formation may be by molding or machining ofthe selected material, with the former being preferred for economy ofmanufacture.

The sidewall of the microvessel 98 is formed of porous or semipermeablenon-reactive material, such as PTFE mesh, preferably having a 70 μM poresize. The sidewall is preferably attached to the top ring 84 and bottomcap 86 of the solid material frame 82. Such attachment may be by knownmeans such as bonding with appropriate glues or other chemicals or heat,with heat being preferred.

In the embodiment of FIGS. 14-16, the two support ribs 88, 100 arepositioned opposite one another, however, any number of support ribs,i.e., one or more, may be provided. The microvessel sidewall 98 need notbe fully attached to the support ribs 88, 100, however, the moldingprocess by which the microvessels are formed may result in attachment atall contact points between the frame and the sidewall.

In the preferred manufacturing process, the sidewall material, a flatsheet of mesh, is rolled into a cylinder and placed inside the mold. Theframe material is injected into the mold around the mesh, causing theframe to fuse to the mesh at all contact points, and sealing the edgesof the mesh to close the cylinder.

In the embodiment illustrated in FIGS. 14-15, the microvessel isconfigured with a removable end cap 90. The end cap 90 is preferablyconstructed of the same material as the solid material frame 82. A snapring, or, as illustrated, projections 92, 94 extend downward from theinside surface of the end cap 90. The projections 92, 94 have a flangewhich mates with a groove 96 formed in the inner wall of top ring 84when pressed into the top ring to releasable secure the end cap 90 tothe microvessel 80. As will be apparent, other means for releasablysecuring the end cap 90 to the top ring 84 can be used, including, butnot limited to, those alternatives stated for the embodiment of FIGS.11-13. The dimensions vary as described for the microvessel of FIGS.11-13 and elsewhere herein.

In other embodiments, these vessels fabricated in any desired orconvenient geometry, such as conical shapes. They can be solid at oneend, and only require a single cap or sealable end.

These microvessels are preferably fabricated as follows. The solidportions, such as the solid cap and body, are fabricated from apolypropylene resin, Moplen resin [e.g., V29G PP resin from Montell,Newark Del., a distributor for Himont, Italy]. The mesh portion isfabricated from a polypropylene, polyester, polyethylene orfluorphore-containing mesh [e.g., PROPYLTEX®, FLUORTEX®, and other suchmeshes, including cat. no. 9-70/22 available from TETKO® Inc, BriarcliffManor, N.Y., which prepares woven screening media, polypropylene mesh,ETF mesh, PTFE mesh, polymers from W.L. Gore. The pores are any suitablesize [typically about 50-100 μM, depending upon the size of theparticulate matrix material] that permits contact with the syntheticcomponents in the medium, but retains the particulate matrix particles.

EXAMPLE 4

Manual System

Illustrated in FIG. 17 is a program/read station for writing to andreading from the memory devices in the microvessel. The electroniccomponents are commercially available from the same supplier of thememory devices, e.g., BMDS or ID TAG [Bracknell Berks RG12 3XQ, UK], sothat the basic operations and frequency are compatible. The basiccontroller 170 and the transceiver 172 are disposed within a housing 174which has a recessed area 176 positioned within the transmission rangeof coil 178. The microvessel 180 may be placed anywhere within recessedarea 176, in any orientation, for both programming and readingfunctions. Basic controller 170 is connected to the system controller182, illustrated here as a functional block, which provides the commandsand encoded data for writing to the memory device in the microvessel andwhich receives and decodes data from the memory device during the readfunction. System controller 182 is typically a PC or lap top computerwhich has been programmed with control software 184 for the variouswrite and read functions.

An example of the operation of the system of FIG. 17 is illustrated inFIG. 18. When power is supplied to the system, transceiver 172 emits aninterrogation signal 185 to test for the presence of a memory device,i.e., a responder, within its detection range. The interrogation signal185 is essentially a read signal that is continuously transmitted untila response 186 is received. The user manually places a microvessel 180within the recessed area 176 so that the interrogation signal 185provides a response to the controllers indicating the presence on themicrovessel. The system receives the interrogation signal and performs adecode operation 187 to determine the data on the memory device withinthe microvessel, which data may include identification of the device anddata concerning prior operations to which the microvessel has beenexposed. Based upon the data obtained, the system makes a determination188 of whether additional information is to be written. The system thenperforms a write operation 189 to record the immediately precedingoperation. The write operation 189 involves modulating the transmittedsignal as a series of “0's” and “1's”, which are recorded on the memorychip, which typically has a 128 bit capacity. After completion of theprogramming step 189, an error check 190 is performed wherein a secondread signal is emitted to verify the data that was written for integrityand correct content. If the correct data is not verified, the system mayattempt to perform the write operation 189 again. After verification ofthe correct data, if the microvessel is one that should proceed toanother operation, the system controller 182 will display instructions192 for direction of the microvessel to the next process step.

The read operation is the same as the beginning of the write operation,with the interrogation signal being continuously transmitted, ortransmitted at regular intervals, until a response is received. Theresponse signal from the memory device in the microvessel 180 isconducted to system controller 182 for decoding and output of the datathat is stored on the memory device. Software within the systemcontroller 182 includes a data base mapping function which provides anindex for identifying the process step associated with data written atone or more locations in the memory device. The system memory within thesystem controller 182 will retain the identification and process stepsfor each microvessel, and an output display of the information relatingto each microvessel can indicate both where the microvessel has been,and where it should go in subsequent steps, if any. After the datastored within the microvessel has been read, it is removed from theinterrogation field and advanced to its next process step.

EXAMPLE 5

Preparation of a Library and Encoding the Matrices with Memories

A pool of the matrices with memories prepared as in EXAMPLE 2 was splitinto two equal groups. Each group was then addressed and write-encodedwith a unique radio frequency signal corresponding to the buildingblock, in this instance an amino acid, to be added to that group.

The matrices with memories were then pooled, and common reactions andmanipulations such as washing and drying, were performed. The pool wasthen re-split and each group was encoded with a second set of radiofrequency signals corresponding to the next set of building blocks to beintroduced, and the reactions were performed accordingly. This processwas repeated until the synthesis was completed. The semiconductordevices also recorded temperature and can be modified to record otherreaction conditions and parameters for each synthetic step for storageand future retrieval.

Ninety-six matrices with memories were used to construct a 24-memberpeptide library using a 3×2×2×2 “split and pool” strategy. Thereactions, standard Fmoc peptide syntheses [see, e.g., Barany et al.(1987) Int. J. Peptide Protein Res. 30:705-739] were carried outseparately with each group. All reactions were performed at ambienttemperature; fmoc deprotection steps were run for 0.5 h; coupling stepswere run for 1 h; and cleavage for 2 h. This number was selected toensure the statistical formation of a 24-member library [see, Burgess etal. (1994) J. Med. Chem. 37:2985].

Each matrix with memory in the 96-member pool was decoded using aspecifically designed radio frequency memory retrieving device [BioMedic Data Systems Inc. DAS-5001 CONSOLE™ System, see, also U.S. Pat.No. 5,252,962 and U.S. Pat. No. 5,262,772] the identity of the peptideon each matrix with memory [Table 2]. The structural identity of eachpeptide was confirmed by mass spectrometry and ¹H NMR spectroscopy. Thecontent of peptide in each crude sample was determined by HPLC to behigher than 90% prior to any purification and could be increased furtherby standard chromatographic techniques. TABLE 2 Radio frequency EncodedCombinatorial 24-member peptide library # of Entry RF matrices with Mass(SEQ ID) code Peptide memories^(a,b) (Actual)^(c) 1 LAGD Leu-Ala-Gly-Asp3 372 (372.2) 2 LEGD Leu-Glu-Gly-Asp 4 432 (432.2) 3 SAGDSer-Ala-Gly-Asp 5 348 (348.1) 4 SEGD Ser-Glu-Gly-Asp 5 406 (406.1) 5LAVD Leu-Ala-Val-Asp 4 416 (416.2) 6 LEVD Leu-Glu-Val-Asp 6 474 (474.2)7 SAVD Ser-Ala-Val-Asp 2 390 (390.2) 8 SEVD Ser-Glu-Val-Asp 3 446(446.2) 9 LAGF Leu-Ala-Gly-Phe 5 406 (406.2) 10 LEGF Leu-Glu-Gly-Phe 5464 (464.2) 11 SAGF Ser-Ala-Gly-Phe 5 380 (380.2) 12 SEGFSer-Glu-Gly-Phe 6 438 (438.2) 13 LAVF Leu-Ala-Val-Phe 6 448 (448.3) 14LEVF Leu-Glu-Val-Phe 2 xxx 15 SAVF Ser-Ala-Val-Phe 2 xxx 16 SEVFSer-Glu-Val-Phe 1 480 (480.2) 17 LAGK Leu-Ala-Gly-Lys 2 387 (387.3) 18LEGK Leu-Glu-Gly-Lys 1 445 (445.3) 19 SAGK Ser-Ala-Gly-Lys 4 361 (361.2)20 SEGK Ser-Glu-Gly-Lys 3 419 (419.2) 21 LAVK Leu-Ala-Val-Lys 4 429(429.3) 22 LEVK Leu-Glu-Val-Lys 6 487 (487.3) 23 SAVK Ser-Ala-Val-Lys 6403 (403.3) 24 SEVK Ser-Glu-Val-Lys 6 461 (461.3)^(a)This is the number of packets of each matrix with memory containingthe same peptide.^(b)The ambient temperature was recorded by the sensor device of thechip in the matrices with memories at various points during thesynthetic pathway.^(c)Mass refers to (M + H) except entry 1 and 8 which refer to (M-H).Since each peptide has a unique mass, the mass spectrum confirms itsstructure.^(d)HPLC conditions: Shimadzu SCL 10A with a MICROSORB-MV ™ C-18 column(5 μM, 100 Å; isocratic elution with acetonitrile/water.

EXAMPLE 6

Synthesis of a Decapeptide Library

Materials and Methods

(1) A memory device [IPTT-100, Bio Medic Data Systems, Inc., Maywood,N.J.], which is 8×1×1 mm, and TENTAGEL® beads (20 mg) were encapsulatedusing a porous membrane wall and sealed (final size≈10×2×2 mm) asdescribed in Example 2. In particular, each memory with matrixmicrovessel 20 mg of TENTAGEL® resin carrying the acid-cleavable linkerPAL.

(2) Solvents and reagents [DMF, DCM, MeOH, Fmoc-amino acids, PyBOP,HATU, DIEA, and other reagents] were used as received. Mass spectra wererecorded on an API I Perkin Elmer SCIEX Mass Spectrometer employingelectrospray sample introduction. HPLC was performed with a Shimadzu SCI10A with an AXXiOM C-18 column [5 μm, 100 Å; gradient: 0-20 min, 25-100%acetonitrile/water (0.1% TFA]. UV spectra were recorded on a ShimadzuUV-1601 instrument. Peptide sequencing was performed using a Beckmanmodel 6300 amino acid analyzer. Chemicals, solvents and reagents wereobtained from the following companies: amino acid derivatives(CalBiochem); solvents (VWR; reagents (Aldrich-Sigma).

(3) General Procedure for Fmoc-Amino Acid Coupling

The matrix with memory microvessels were placed in a flat-bottomedflask. Enough DMF [v_(r) ml, 0.75 ml per microvessel] was added tocompletely cover all the matrix with memory microvessels. Fmoc-aminoacid, EDIA, and PyBOP [or HATU for the hindered amino acids Pro and Ile]were added sequentially with final concentrations of 0.1, 0.2, and 0.1M, respectively. The flask was sealed and shaken gently at ambienttemperature for 1 h. The solution was removed and the matrix with memorymicrovessels were washed with DMF [4×v_(r)], and resubjected to the samecoupling conditions with half the amount of reagents. They ere finallywashed with DMF [4×v_(r)], MeOH [4×v₄], DCM [4×v₄], and dried undervacuum at ambient temperature.

(4) Fmoc-Deprotection

The matrix with memory microvessels were placed in a flat bottomedflash. Enough 20% piperidine solution in DMF [v_(r) ml, 0.75 ml/matrixwith memory microvessel] was added to completely cover the microvessels.The flask was sealed and gently shaken at ambient temperature for 30min. Aliquots were removed and the UV absorption of the solution wasmeasured at 302 nm to determine the Fmoc number. The matrix with memorymicrovessels were then washed with DMF [6×v_(r)] and DCM [6×v_(r)] anddried under vacuum at ambient temperature.

(5) Procedure for Peptide Cleavage from Solid Support

The TENTAGEL® beads [20-120 mg] from each matrix with memory microvesselwere treated with 1 ml of TFA cleavage mixture[EDT:thioanisole;H₂O:PhOH:TFA, 1.5:3:3:4.5:88, w/w] at ambienttemperature for 1.5 hours. The resin beads were removed by filtrationthrough a glass-wool plug, the solution was concentrated, diluted withwater [2 ml], extracted with diethyl ether [8×2 ml], and lyophilized toyield the peptide as a white powder [4-20 mg].

(6) Preparation of Polyclonal Antibodies

The peptide (SEQUENCE ID No. 25) with a cysteine at the N-terminus, wassynthesized by standard solid phase methods using an automated AppliedBiosystems 430A peptide synthesizer [see, Sakakibara (1971) Chemistryand Biochemistry of Amino Acids, Peptides and Proteins, Weinstein, ed,Vol. 1, Marcel Dekker, NY, pp. 51-85]. The synthetic peptide wasconjugated to keyhole limpet hemocyanin usingmaleimidohexanoyl-N-hydroxysuccinimide as a cross-linking agent [see,Ishikawa et al. (1983) J. Immunoassay 4:209-237]. Rabbits were injectedat multiple dorsal intradermal sites with 500 μg peptide emulsified withcomplete Freund's adjuvant. The animals were boosted regularly at 3-6week intervals with 200 μg of peptide conjugate emulsified in incompleteFreund's adjuvant. The titer of the antisera after a few boosterinjections was approximately 1:50,000 to 1:100,000 as determined byELISA using the unconjugated peptide as the antigen.

(7) Enzyme Linked Immunosorbant Assay [ELISA]

Plates were coated with 100 μl/well of a 0.5 μg/l solution of peptidesdiluted in phosphate buffered saline [PBS] by incubating them overnightat 4° C. The plates were washed extensively with PBS and incubated with200 μl of 0.1% bovine serum albumin [BSA] in PBS for 1 h at roomtemperature. The plates were then washed with PBS and 100 μl of prebledor rabbit anti-peptide [peptide of SEQ ID No. 25] antibody [1:100,000]was added to the duplicate wells. After a 1 h incubation at ambienttemperature, the plates were washed with PBS and 100 μl ofperoxidase-goat-antirabbit IgG diluted in PBS supplemented with 0.1% BSAwas added. After incubation for another hour at ambient temperature, theplates were extensively washed with PBS and 100 μl of peroxidasesubstrate solution was added to each well. The plates were thenincubated for 15 minutes at ambient temperature. The peroxidase reactionwas measured by the increase in absorbance at 405 nm.

The Library

The library included the peptide having the sequenceMet-Leu-Asp-Ser-Ile-Trp-Lys-Pro-Asp-Leu [MLDSIWKPDL; SEQ ID NO. 25],against which an antibody had been generated in rabbits [the peptideused for rabbits had an additional N-terminal Cys residue for linking],and seven other peptides differing at residues L, P, and/or I [SEQ IDNOs. 26-32 and the Scheme set forth in FIG. 10].

The matrix with memory microvessels loaded with TENTAGEL® beads carryingPAL linkers [20 mg each] were split into two equal groups. Each groupwas encoded with the radio frequency code L or A [the one-letter symbolsfor amino acids leucine and alanine, respectively] and the firstcoupling was carried out separately using Fmoc-Leu-OH or Fmoc-Ala-OH,respectively and ByBOP, or HATU for the sterically hindered amino acids[STEP 1, FIG. 10]. The microvessels were then pooled, deprotected with20% piperidine in DMF [Fmoc removal], encoded with the code D andsubjected to coupling with Fmoc-Asp(OtBu)-OH and deprotection as above[STEP 2]. The microvessels were then re-split into two equal and fullyrandomized groups and encoding was performed on each group with thecodes P or F and amino acid derivatives Fmoc-Pro-OH or Fmoc-Phe-OH werecoupled, respectively [STEP 3]. The microvessels were pooled again andamino acid derivatives Fmoc-Lys(Boc)-OH and Fmoc-Trp(Boc)-OH werecoupled sequentially with appropriate encoding and deprotectionprocedures [STEPS 4 and 5], and then were re-split into two equalgroups, encoded appropriately and the amino acid derivatives Fmoc-Ile-OHor Fmoc-Gly-OH were coupled separately [STEP 6]. The matrix with memorymicrovessels were pooled, the amino groups deprotected and the remainingamino acids [Ser, Asp, Leu and Met] were sequentially introduced withappropriate encoding and deprotections using suitably protected Fmocderivatives [STEPS 7-10]. The introduction of each amino acid wasperformed by double couplings at every step. The coupling efficiency foreach step was generally over 90% as measured by Fmoc numberdetermination after Fmoc deprotection [UV spectroscopy].

Decoding each matrix with memory allowed identification of identicalunits. It was observed that a fairly even distribution of matrix withmemories over the entire library space was obtained. It should be notedthat sorting out the matrices with memories at each split by decodingallows this random process to become an exact, “one compound-one matrixwith memory method.

TENTAGEL® beads from matrices with memories with identical codes werepooled together and the peptides were cleaved from the resin separatelywith EDT:thioanisole:H₂O:PhOH:TFA [1.5:3:3:4.5:88m, w/w]. The work-upand isolation procedures involved filtration, evaporation, dilution withwater, thorough extraction with diethyl ether, and lyophilization. Thefully deprotected peptides were obtained as white solids, theirstructures were confirmed by mass spectroscopy, and their purity wasdetermined by HPLC analysis. The peptide sequence in entry 2, [SEQ IDNO. 26] was confirmed by peptide amino acid sequence analysis. Ambientreactor temperature was also measured at specific synthesis steps by theon-board temperature thermistor.

Biological Screening of the Peptide Library

A rabbit polyclonal antibody generated specifically against the peptideSEQ ID NO. 25 was used to detect this specific sequence in the REC™peptide library by the ELISA method. The ELISA assay correctlyidentified the library member with the SEQ ID NO. 25 [100% binding]. Thesequence of this peptide was also confirmed by the radio frequency code,mass spectroscopy, and amino acid sequence analysis.

It was also of interest to observe trends in the binding of the antibodyto the other members of the library. It was observed that the binding ofeach peptide was dependent on the type, position, and number ofmodifications from the parent sequence. Thus, replacement of I with Gdid not change significantly the antigenicity of the peptide.Substitution of L with A reduced antibody binding by ≈40% andreplacement of P with F essentially converted a peptide to anon-recognizable sequence. Replacement of two amino acids resulted insignificant loss of binding. Thus the concurrent substitutions [I→G andP→F], [I→G and L→A], and [P→F and L→A] reduced antibody binding by ≈40,60, and 92%, respectively. Finally, the peptide library member in whichI, P and L were replaced with G, F and A, respectively, was notrecognized by the antibody. Collectively, these results suggest thatamino acids at the C-terminus of the peptide, especially P play animportant role in this particular antibody-peptide recognition.

EXAMPLE 7

Procedures for Coating Glass-Enclosed Memory Devices with SilylatedPolystyrene

A procedure for coating glass-enclosed memory devices, such as theIPTT-100, is represented schematically as follows:

A. Procedure A

1. Before coating, the glass surface of the IPTT-100 transponder wascleaned using base, chloroform, ethanol and water, sequentially, and,then heated to 200° C. [or 300° C.] to remove water.

2. The residue from the solvents in step 1 were removed under vacuum.

3. N-styrylethyltrimethoxy silane HCl, chloromethyl styrene, divinylbenzene and benzoyl peroxide [9:1:0.1:0.2 mol] is stirred for 10minutes.

4. The resulting mixture was coated on the cleaned glass, which was thenbaked at 150-200° C. for 5 to 10 minutes in air or under nitrogen.

5. The coated glass was then sequentially washed with DCM, DMF andwater. The resulting coating was stable in DCM, DMF, acid and base forat two weeks at 70° C.

B. Procedure B

1. Before coating, the glass surface is cleaned using base, chloroform,ethanol and water, sequentially, and, then heating to 200° C. [or 300°C.] to remove water.

2. The residue from the solvents in step 1 are removed under vacuum.

3. N-styrylethyltrimethoxy silane HCl [10-15%] is refluxed in toluenewith the cleaned glass surface.

4. After reaction, the glass surface is washed with toluene, DCM,ethanol and water sequentially.

5. A mixture of chloromethyl styrene, divinyl benzene and benzoylperoxide [molar ratio of N-styrylethyltrimethoxy silane HCl to the othercompounds is 9:1:0.1:0.2 mol] is coated on the glass, which is thenbaked at 150-200° C. for 10 to 60 minutes.

6. The coated glass is then sequentially washed with toluene, DCM, DMFand water.

EXAMPLE 8

Preparation of Scintillant-Encased Glass Beads and Chips

Materials:

POPOP [Aldrich] or PPO [concentrations about 5 to 6 g/l], and/orp-bis-o-methylstyrylbenzene [bis-MSB] or di-phenylanthracene [DPA][concentrations about 1 g/l], or scintillation wax [FlexiScint fromPackard]. Precise concentrations may be determined empirically dependingupon the selected mixture of components.

Porous glass beads [Sigma]

IPTT-100 transponders [see, Examples 2-4].

A. Preparation of Scintillant Coated Beads

Porous glass beads are soaked in a mixture of PPO [22-25% by weight] andbis-MSB [up to 1% by weight] in a monomer solution, such styrene orvinyltoluene, or in hot liquified scintillation wax [3-5 volume/volumeof bead]. A layer of polystyrene [about 2 to 4 μM] is then applied. Apeptide is either synthesized on the polystyrene, as described above, oris coated [adsorbed] or linked via a cleavable linker to thepolystyrene.

B. Preparation of Scintillant Coated Matrix with Memory Beads

1. The porous glass beads are replaced with glass encased [etched priorto use] transponders and are treated as in A. The resulting beads aresealed with polystyrene [2 to 5 μM] and then coated with a selectedacceptor molecule, such as an antigen, antibody or receptor, to which aradiolabeled ligand or antibody selectively binds. The identity of thelinked peptides or protein is encoded into each memory. After reactionand counting in a liquid scintillation counter, the beads that havebound acceptor molecule are read to identify the linked protein.

2. The porous glass beads are replaced with glass encased [etched priorto use] transponders and are treated as in A and sealed as in A withpolystyrene. A peptide, small organic or other library is synthesized onthe polystyrene surface of each bead, and the identity of each member ofthe library encoded into the memory. The beads with linked molecules arereacted with labeled receptor and counted in a liquid scintillationcounter. After counting in a liquid scintillation counter, the beadsthat have bound receptor are read to identify the molecule that bound tothe receptor.

EXAMPLE 9

Use of the Scintillant Coated or Encased Particles in Assays

In experiments 1-3, as model system, the binding of biotin to functionalamine groups was detected using ¹²⁵I-strepavidin. In experiment 4, thebinding of [Met⁵] enkephalin to the functional amine groups was detectedusing ¹²⁵I-antibody.

Experiment #1

1. Scintillant [PPO %2 and DPA %0.05] was introduced [EmeraldDiagnostics, Eugene, Oreg.] and incorporated on the interior surface ofpolystyrene beads [Bang Laboratories]. The polystyrene beads were 3.1μM, with 20% crosslinking and were derivatized with amine groups.

2. The concentration of the functional amine groups on the bead surfaceswas estimated to be about 0.04125 μmol/mg. The amine groups werecovalently linked to the N-hydroxy succinimide derivative of Biotin[Calbiochem 203112] at molecular ratio of 1:10, respectively. This wasdone by resuspending the beads in a 50% acetonitrile: water, Hepes [pH8.0] buffered solution containing biotin for 2 hours at roomtemperature. After 2 hours, the beads were washed 6 times with 10 ml of50% acetonitrile in water. Beads were resuspended in PBS [pH 7.2] andstored overnight at 4° C.

3. Using an SPA format, biotin was detected using ¹²⁵I-streptavidin tothe biotin was detected. This was done by diluting beads to a 20 mg/mland adding them to 96 well plates at 4, 2, 1. 0.5, 0.25, and 0.125 mgper well. Volumes were adjusted to 100 ul per well. ¹²⁵I-strepavidin wasadded to final concentration of 0.1 μCi per well. Plates were counted ina Wallac MicroBeta Trilux scintillation counter after 2 hours. Boundbiotin was detected.

Experiment #2

1. Scintillants [pyrenebutyric acid and 9-anthracenepropionic acid] werecovalently linked to the TENTAGEL® beads, with 0.25 mmol/g availablefunctional amine groups, at 2%: 0.05% ratio, respectively. Thefluorophore was linked to 15% of these sites.

2. The functional amine group on the TENTAGEL® beads were covalentlylinked to the N-hydroxy succinimide derivative of biotin. The freefunctional amine groups on beads [0.21 μmol/mg] were covalently linkedto biotin [Calbiochem 203112]. Briefly, Biotin was mixed with the beadsat a molecular ratio of 10:1 in 6 ml of 50% acetonitrile with Hepes [pH8.0] and incubated for 2 hours at room temperature. At the end ofincubation period, the beads were washed 3 times with 10 ml of 100%acetonitrile followed by 3 washes with 50% acetonitrile in water. Thebeads were resuspended in PBS [pH 7.2] and stored overnight at 4° C.

3. Biotin was detected using ¹²⁵I-streptavidin detected in a SPA format.This was done by diluting beads to a 20 mg/ml, and introducing them intowells in 96 well plats at 4, 2, 1, 0.5, 0.25, and 0.125 mg per well.Volumes were adjusted to 100 μl per well. ¹²⁵I-streptavidin [AmershamIM236] was added to each well at a concentration of 0.05 μCi/well. Afterapproximately 2 hours, additional ¹²⁵I-strepavidin was added for a finalconcentration of 0.1 uCi per well. Plates were counted in a WallacMicroBeta Trilux scintillation counter after 2 hours. Bound biotin wasdetected.

Experiment #3

1. BMDS chips [and also similar chips ID TAG available fromIdentification Technologies Inc.] were coated with scintillant [PPO %2and DPA 0.5% in polystyrene [10% in dichloromethane].

2. The chip was then coated with a layer of derivatized silane.

3. The functional amine groups were covalently linked to the N-hydroxysuccinimide derivative of Biotin. The free functional amine groups onthe silane [375 nmol/chip] were covalently linked to Biotin [Calbiochem203112]. Briefly, biotin was dissolved in 1 ml of 30% acetonitrile withHepes [pH 8.0] and incubated with the chip for 2 hours at roomtemperature. At the end of incubation period, the chip washed 3 timeswith 50% acetonitrile in water, resuspended in PBS [pH 7.2] and storedovernight at 4° C.

4. Biotin was detected in a SPA format by ¹²⁵I-streptavidin. The chipswere placed in 24-well plate with 500 μl ¹²⁵I-streptavidin [0.1μCi/well, Amersham IM236]). After a 2 hour incubation, the plates werecounted in Wallac MicroBeta Trilux scintillation counter. Binding wasdetected.

Experiment #4

1. The chips were coated with scintillant [PPO %2 and DPA] 0.05% inpolystyrene [10% in dichloromethane].

2. The functional amine group was derivatized for spontaneous covalentbinding to amine group [Xenopore, N.J.].

3. [Met⁵]Enkephalin [tyr-gly-gly-phe-met; SEQ ID No. 33] peptide [R&DAntibodies] were covalently linked to the amine group by incubating thecoagted chip with the peptide in 500 μl of PBS [160 μg peptide/ml, pH8]) overnight at room temperature.

4. At the end of the incubation, the chips were washed and thenincubated in 3% bovine serum albumin for 2 hours.

5. Linked peptide was detected in a SPA format. The chips were wasplaced in 24-well plate containing 500 μl of ¹²⁵I-anti-[Met5]Enkephalinantibody [0.1 μCi/well, R&D Antibodies]. The antibody is a rabbitpolyclonal against the C-terminal region of the peptide. After a 2 hourincubation, the plates were counted in Wallac MicroBeta Triluxscintillation counter and linked peptide was detected.

Since modifications will be apparent to those of skill in this art, itis intended that this invention be limited only by the scope of theappended claims.

1. A combination of a matrix with memory, comprising: a memory devicecomprising an electromagnetically-readable memory containing encodeddata; and a solid support matrix material, wherein the matrix materialis comprised of particles of a size such that at least one dimension isno more than about 10 mm; wherein the encoded data uniquely identifiesthe molecule or biological particle linked to the solid support matrix.2-5. (canceled)
 6. The combination of claim 1, wherein theelectromagnetically-readable memory is an alphanumeric code or a barcode. 7-18. (canceled)
 19. The combination of claim 1, furthercomprising luminescent moieties associated with the matrix material.20-52. (canceled)
 53. The combination of claim 1, wherein the moleculeis a protein, peptide, or nucleic acid.
 54. The combination of claim 1,wherein the biological particle is a phage, prokaryotic cell oreukaryotic cell. 55-163. (canceled)
 164. A multiplexed cell-based assay,comprising: attaching each cell of a plurality of cells to a supportmatrix adapted for attachment and/or growth of cells, wherein thesupport matrix is physically linked to a memory device, the memorydevice comprising an electromagnetically-readable memory containingencoded data to uniquely identify a cell attached to the support matrix;exposing the plurality of cells to one or more processing conditions;placing the plurality of support matrices with the attached cells into acommon vessel; and measuring cellular biochemical processes within thecommon vessel; wherein each of the plurality of supports has differentencoded data in its linked memory device so that when the plurality ofsupports are placed into the common vessel, the cell associated witheach support has a unique identity associated therewith.
 165. The assayof claim 164, wherein the plurality of cells is selected from the groupconsisting of established cell lines, primary cell cultures, reportergene systems in recombinant cells, cells transfected with a selectedgene, and recombinant mammalian cells.
 166. The assay of claim 164,wherein the support matrix is formed from a plastic that is opticallytransparent.
 167. The assay of claim 166, wherein the support matrix isformed from a plastic having a scintillant incorporated therein. 168.The assay of claim 164, wherein the electromagnetically-readable memoryis an alphanumeric code or a bar code disposed on the support matrix anda remote memory stores the encoded data in association with informationabout the cell attached to the support matrix.
 169. The assay of claim164, wherein the one or more processing conditions comprises culturingthe cells.
 170. The assay of claim 164, wherein the one or moreprocessing conditions comprises labeling the cells.
 171. The assay ofclaim 164, wherein the one or more processing conditions comprisesexposing the cells to a compound.
 172. The assay of claim 164, whereinmeasuring comprises detecting fluorescence.
 173. In an assay forevaluating a plurality of molecules or biological particles, whereineach of the molecules or biological particles is linked to a separatesupport matrix, the improvement comprising combining the support matrixwith an electromagnetically-readable memory containing encoded data touniquely identify the molecule or biological particle linked to thesupport matrix.
 174. The assay of claim 173, wherein the support matrixis formed from a plastic that is optically transparent.
 175. The assayof claim 174, wherein the support matrix is formed from a plastic havinga scintillant incorporated therein.
 176. The assay of claim 173, whereinthe electromagnetically-readable memory is an alphanumeric code or a barcode.
 177. The assay of claim 173, wherein the biological particles areselected from the group consisting of viruses, phages, cells, cellfragments, liposomes, and micellar agents.
 178. The assay of claim 173,wherein the molecules are selected from the group consisting ofproteins, peptides, nucleic acids, bio-oligomers, amino acids, sequencesof random monomer subunits, and polymers of small organic molecularconstituents of non-peptidic libraries.